US20160096869A1 - Chlorotoxin conjugates and methods of use thereof - Google Patents

Chlorotoxin conjugates and methods of use thereof Download PDF

Info

Publication number
US20160096869A1
US20160096869A1 US14/855,355 US201514855355A US2016096869A1 US 20160096869 A1 US20160096869 A1 US 20160096869A1 US 201514855355 A US201514855355 A US 201514855355A US 2016096869 A1 US2016096869 A1 US 2016096869A1
Authority
US
United States
Prior art keywords
alkylene
compound
hydrogen
cit
cancer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US14/855,355
Other languages
English (en)
Inventor
Stacey J. Hansen
Claudia Jochheim
Dennis M. Miller
Natalie Nairn
Julia E. Novak
Mark Stroud
Valorie R. Wiss
Kelly Byrnes-Blake
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Blaze Bioscience Inc
Original Assignee
Blaze Bioscience Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Blaze Bioscience Inc filed Critical Blaze Bioscience Inc
Priority to US14/855,355 priority Critical patent/US20160096869A1/en
Assigned to Blaze Bioscience, Inc. reassignment Blaze Bioscience, Inc. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WISS, VALORIE R., JOCHHEIM, CLAUDIA, HANSEN, STACEY J., MILLER, DENNIS M., NAIRN, Natalie, NOVAK, JULIA E., STROUD, MARK, BYRNES-BLAKE, KELLY
Publication of US20160096869A1 publication Critical patent/US20160096869A1/en
Assigned to Blaze Bioscience, Inc. reassignment Blaze Bioscience, Inc. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WISS, VALORIE R., MILLER, DENNIS M., BYRNES-BLAKE, KELLY, HANSEN, STACEY J., JOCHHEIM, CLAUDIA, NAIRN, Natalie, NOVAK, JULIA E., STROUD, MARK
Priority to US16/704,955 priority patent/US20200188536A1/en
Priority to US17/409,661 priority patent/US11559580B1/en
Priority to US17/888,072 priority patent/US20230211000A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43522—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from scorpions
    • A61K47/48261—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • compounds of the present disclosure have the structure of Formula (I), or a pharmaceutically acceptable salt thereof:
  • the presently described compounds further comprise a detectable label, which can be used for the detection of the peptide-label conjugate and the cancerous cells to which they are bound.
  • compounds of the present disclosure have the structure of Formula (XV), or a pharmaceutically acceptable salt thereof:
  • the compounds of the present disclosure have a structure of Formula (II), or a pharmaceutically acceptable salt thereof:
  • the present compounds have a structure of Formula (III), or a pharmaceutically acceptable salt thereof:
  • compounds of the present disclosure have a structure of Formula (IV), or a pharmaceutically acceptable salt thereof:
  • compounds of the present disclosure have a structure of Formula (V), or a pharmaceutically acceptable salt thereof:
  • compounds of the present disclosure have a structure of Formula (VI), or a pharmaceutically acceptable salt thereof:
  • compounds of the present disclosure have a structure Formula (III), or a pharmaceutically acceptable salt thereof:
  • the present disclosure provides a kit comprising vessel configured to contain a fluid; any of the compounds and compositions described herein; and an elastomeric closure affixed to the vessel.
  • the present disclosure provides a composition
  • a composition comprising a compound comprising a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof, wherein when the composition is intravenously administering to a human subject at a dose of from 1 mg to 30 mg, the composition produces in the human subject an average maximum compound blood plasma concentration (average C max ) of at least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of the compound administered.
  • average C max average maximum compound blood plasma concentration
  • the present disclosure provides a method of administering a composition to a human subject, the method comprising: intravenously administering to the human subject a dose of from 1 mg to 30 mg of a compound comprising a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof; and producing in the human subject an average maximum compound blood plasma concentration (average C max ) of at least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of the compound administered.
  • average C max average maximum compound blood plasma concentration
  • the present disclosure provides a method of detecting a cancer cell in a human subject, the method comprising: intravenously administering to the human subject a dose of from 1 mg to 30 mg of a compound comprising a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof conjugated to a detectable label; producing in the human subject an average maximum compound blood plasma concentration (average C max ) of at least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of the compound administered; and detecting the presence or absence of the detectable label in the human subject, wherein the presence of the detectable label indicates the presence of the cancer cell.
  • average C max average maximum compound blood plasma concentration
  • the present disclosure provides a method of diagnosing cancer in a human subject, the method comprising: intravenously administering to the human subject a dose of from 1 mg to 30 mg of a compound comprising a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof conjugated to a detectable label; producing in the human subject an average maximum compound blood plasma concentration (average C max ) of at least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of the compound administered; and detecting the presence or absence of the detectable label in the human subject, wherein the presence of the detectable label indicates a diagnosis of cancer.
  • the present disclosure provides a method of treating cancer in a human subject, the method comprising: intravenously administering to the human subject a dose of from 1 mg to 30 mg of a compound comprising a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof conjugated to a therapeutic agent; producing in the human subject an average maximum compound blood plasma concentration (average C max ) of at least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of the compound administered; and reducing or improving a symptom or condition associated with cancer in the human subject.
  • the human subject is in need thereof.
  • the methods comprise administering a therapeutically effective dose of the compound to the human subject.
  • the present disclosure provides a method of administering a composition to a human subject, the method comprising: administering to the human subject a dose of from 1 mg to 30 mg of a compound comprising a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof; and producing in the human subject pharmacokinetic profile of FIG. 27 .
  • the present disclosure provides a method of administering a composition to a human subject, the method comprising: intravenously administering to the human subject a dose of from 1 mg to 30 mg of any suitable compound of the present disclosure; and producing in the human subject an average maximum compound blood plasma concentration (average C max ) of at least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of the compound administered.
  • average C max average maximum compound blood plasma concentration
  • the present disclosure provides a method for detecting a cancer cell in a subject, the method comprising: administering any suitable compound of the present disclosure; and detecting the presence or absence of the compound in the subject, wherein the presence of the compound indicates the presence of a cancer cell.
  • the present disclosure provides a method of administering any suitable compound of the present disclosure to a subject, the method comprising administering a therapeutically effective amount of the compound to the subject.
  • the present disclosure provides a method of treating a subject in need thereof, the method comprising administering to the subject any suitable compound of the present disclosure further comprising a therapeutic agent in an amount sufficient to treat cancer in the subject.
  • the therapeutic agent is a cytotoxic agent.
  • the chlorotoxin conjugate comprises a chemotherapeutic, an anti-cancer agent, or an anti-cancer drug.
  • the chlorotoxin conjugate comprising the chemotherapeutic, an anti-cancer agent, or an anti-cancer drug is administered after the central or primary tumor is detected during surgery.
  • the central or primary tumor is detected with a chlorotoxin conjugated to a labeling agent.
  • a method of inhibiting, preventing, minimizing, shrinking, or killing cells, or preventing metastasis in residual tumor cells in a tumor bed in an individual comprising the step of administering a chlorotoxin conjugate to the individual wherein the chlorotoxin conjugate binds to the residual tumor cells in a tumor bed; and whereby the residual tumor cells in the tumor bed are inhibited, prevented, minimized, shrinked, or killed cells, or metastasis is prevented.
  • the chlorotoxin conjugate comprises a chemotherapeutic, an anti-cancer agent, or an anti-cancer drug.
  • the chlorotoxin conjugate comprising the chemotherapeutic, an anti-cancer agent, or an anti-cancer drug is administered after the central or primary tumor is detected during surgery.
  • the central or primary tumor is detected with a chlorotoxin conjugated to a labeling agent.
  • the invention provides, a method of administering a chlorotoxin conjugated to a chemotherapeutic, an anti-cancer agent, or an anti-cancer drug to an individual to treat, inhibit, prevent, minimize, shrink, or kill cells, or prevent metastasis in cells that are identified with a chlorotoxin conjugated to a labeling agent.
  • the anti-cancer agent include antibodies, polypeptides, polysaccharides, and nucleic acids.
  • the chlorotoxin conjugate is administered about 1 day before the surgery. In another embodiment, the chlorotoxin conjugate is administered about 2 days before surgery. In another embodiment, the chlorotoxin conjugate is administered in multiple sub-doses.
  • the chlorotoxin conjugate is administered in about 2 sub-doses, 3 sub-doses, 4 sub-doses, or more sub-doses.
  • the chlorotoxin conjugate comprising the chemotherapeutic, an anti-cancer agent, or an anti-cancer drug is administered after the central or primary tumor is detected during surgery.
  • the central or primary tumor is detected with a chlorotoxin conjugated to a labeling agent.
  • the invention provides a method of detecting soft-tissue sarcoma in an individual, comprising the steps of: a) administering a chlorotoxin conjugate to the individual wherein the chlorotoxin conjugate binds to the soft-tissue sarcoma; and b) imaging, visualizing, or analyzing the bound chlorotoxin conjugate.
  • the detecting comprises in vivo, or ex vivo detecting.
  • the imaging, visualizing, or analyzing comprises visualizing the chlorotoxin conjugate.
  • the imaging, visualizing, or analyzing comprises in vivo, or ex vivo imaging, visualizing, or analyzing.
  • the visualizing comprises optically imaging the sarcoma.
  • a plot is made of the fluorescent intensity of the chlorotoxin conjugate.
  • a chlorotoxin conjugate comprising one or more labeling agents is used in detecting the soft-tissue sarcoma.
  • the labeling agent comprises a fluorescent moiety.
  • the fluorescent moiety comprises a near infrared fluorescent moiety.
  • the labeling agent comprises a radionuclide.
  • the soft-tissue sarcoma is selected from the group consisting of: trachea, fat tissue tumors, muscle tissue tumors, skeletal muscle sarcomas, rhabdomyosarcomas, peripheral nerve tumors, fibrous tissue tumors, myxofibrosarcomas, fibromatosis, joint tissue tumors, tumors of blood vessels and lymph vessels, angiosarcomas, gastrointestinal stromal tumors, alveolar soft part sarcoma, dermatofibrosarcomaprotuberans (DFSP), desmoplastic small round cell tumour, epithelioid sarcoma, extra skeletal myxoid chondrosarcoma, and giant cell fibroblastoma (GCF).
  • trachea fat tissue tumors, muscle tissue tumors, skeletal muscle sarcomas, rhabdomyosarcomas, peripheral nerve tumors, fibrous tissue tumors, myxofibrosarcomas, fibromatosis, joint
  • a chlorotoxin conjugate is used to detect soft-tissue sarcoma in subcutaneous fatty tissue.
  • the detecting comprises imaging, visualizing, or analyzing the chlorotoxin conjugate during or related to surgery, surgical resection, or intraoperative imaging and resection.
  • the sarcoma, or a portion thereof is removed during or related to surgery.
  • the invention provides a method of detecting cutaneous squamous cell carcinoma in an individual, comprising the steps of: a) administering a chlorotoxin conjugate to the individual, wherein the chlorotoxin conjugate binds to the cutaneous squamous cell carcinoma; and b) imaging, visualizing, or analyzing the bound chlorotoxin conjugate.
  • the detecting comprises in vivo, or ex vivo detection.
  • the imaging, visualizing, or analyzing comprises visualizing the chlorotoxin conjugate.
  • the imaging, visualizing, or analyzing comprises in vivo, or ex vivo imaging, visualizing, or analyzing.
  • the invention provides a method of using a chlorotoxin conjugate to optically image cutaneous squamous cell carcinoma.
  • the chlorotoxin conjugate comprises one or more labeling agents.
  • the labeling agent comprises a fluorescent moiety.
  • the fluorescent moiety comprises a near infrared fluorescent moiety.
  • the labeling agent comprises a radionuclide.
  • the detecting comprises imaging, visualizing, or analyzing the chlorotoxin conjugate during or related to surgery, surgical resection, or intraoperative imaging and resection.
  • the cutaneous squamous cell carcinoma, or a portion thereof is removed during or related to surgery.
  • the fluorescent intensity of the chlorotoxin conjugate is made.
  • the invention provides a method of detecting a low-grade tumor in an individual, comprising the steps of: a) administering a chlorotoxin conjugate to the individual wherein the chlorotoxin conjugate binds to the low-grade tumor; and b) imaging, visualizing, or analyzing the bound chlorotoxin conjugate.
  • the detecting comprises in vivo, or ex vivo detecting.
  • the imaging, visualizing, or analyzing comprises vivo, or ex vivo imaging, visualizing, or analyzing.
  • the invention provides a method of using a chlorotoxin conjugate to optically image a low-grade tumor.
  • the chlorotoxin conjugate comprises one or more labeling agents.
  • the labeling agent comprises a fluorescent moiety.
  • the fluorescent moiety comprises a near infrared fluorescent moiety.
  • the labeling agent comprises a radionuclide.
  • the detecting is performed during or related to surgery or resection.
  • the low-grade tumor, or a portion thereof is removed during or related to surgery, surgical resection, or intraoperative imaging and resection.
  • the low-grade tumor is selected from the group consisting of a) a low-grade tumor in or from brain tissue; b) a low-grade tumor in or from subcutaneous fatty tissue; c) a low-grade tumor in or from breast or mammary tissue, and d) a low-grade tumor in or from lung tissue.
  • a plot is made of the fluorescent intensity of the chlorotoxin conjugate.
  • the chlorotoxin conjugate comprises one or more labeling agents.
  • the labeling agent comprises a fluorescent moiety.
  • the fluorescent moiety comprises a near infrared fluorescent moiety.
  • the labeling agent comprises a radionuclide.
  • the detecting comprises in vivo, or ex vivo detection.
  • the imaging, visualizing, or analyzing comprises in vivo, or ex vivo imaging, visualizing, or analyzing.
  • a plot is made of the fluorescent intensity of the chlorotoxin conjugate.
  • a method for using a chlorotoxin conjugate to detect residual cancer in the tumor bed of an individual following removal of a primary or central tumor in breast cancer surgery comprising: a) administering a chlorotoxin conjugate to the individual wherein the chlorotoxin conjugate binds to residual cancer; and b) imaging, visualizing, or analyzing the bound chlorotoxin conjugate.
  • the chlorotoxin conjugate comprises one or more labeling agents.
  • the labeling agent comprises a fluorescent moiety.
  • the fluorescent moiety comprises a near infrared fluorescent moiety.
  • the labeling agent comprises a radionuclide.
  • the detecting is performed during or related to surgery or resection.
  • the residual cancer, or a portion thereof is removed during or related to surgery, surgical resection, or intraoperative imaging and resection.
  • the chlorotoxin conjugate comprises one or more labeling agents.
  • the labeling agent comprises a fluorescent moiety.
  • the fluorescent moiety comprises a near infrared fluorescent moiety.
  • the labeling agent comprises a radionuclide.
  • the invention provides, a method for detecting a tumor in an individual comprising the steps of: a) administering a chlorotoxin conjugate to the individual wherein the chlorotoxin conjugate binds to the tumor; and b) imaging, visualizing, or analyzing the bound chlorotoxin conjugate wherein the chlorotoxin conjugate is administered in an amount of between about 0.9 mg/m 2 to about 1.1 mg/m 2 or in an amount of between about 3 mg to about 6 mg.
  • the detecting comprises vivo, or ex vivo detection.
  • the imaging, visualizing, or analyzing comprises in vivo, or ex vivo imaging, visualizing, or analyzing.
  • the chlorotoxin conjugate comprises one or more labeling agents.
  • the labeling agent comprises a fluorescent moiety.
  • the fluorescent moiety comprises a near infrared fluorescent moiety.
  • the labeling agent comprises a radionuclide.
  • the detecting is performed during or related to surgery or resection.
  • the tumor, or a portion thereof is removed during or related to surgery surgical resection, or intraoperative imaging and resection.
  • the invention provides methods of administering a chlorotoxin conjugate to an individual to detect soft-tissue sarcoma, low-grade tumor, cutaneous squamous cell carcinoma, or cells therefrom, in tumors of skin or breast, and lung and mammary cancers.
  • the chlorotoxin conjugate comprises one or more labeling agents.
  • the labeling agent comprises a fluorescent moiety.
  • the fluorescent moiety comprises a near infrared fluorescent moiety.
  • the labeling agent comprises a radionuclide.
  • the detecting is performed during or related to surgery or resection.
  • the soft-tissue sarcoma, low-grade tumor, cutaneous squamous cell carcinoma, or cells therefrom, in tumors of skin or breast, and lung and mammary cancers, or a portion thereof, is removed during or related to surgery, surgical resection, or intraoperative imaging and resection.
  • the chlorotoxin conjugate is administered in an amount of between about 0.9 mg/m 2 to about 1.1 mg/m 2 or in an amount of between about 3 mg to about 6 mg.
  • the chlorotoxin conjugate is administered about 1 day before the surgery. In another embodiment, the chlorotoxin conjugate is administered about 2 days before surgery.
  • the chlorotoxin conjugate is administered in multiple sub-doses. In an embodiment, the chlorotoxin conjugate is administered in about 2 sub-doses, 3 sub-doses, 4 sub-doses, or more sub-doses.
  • the detecting comprises in vivo, or ex viva detection. In some embodiments, the imaging, visualizing, or analyzing comprises in vivo, or ex vivo imaging, visualizing, or analyzing. In some embodiments a plot is made of the fluorescent intensity of the chlorotoxin conjugate.
  • a method for using a chlorotoxin conjugate to detect residual cancer in the tumor bed of an individual following removal of a primary or central tumor in breast cancer surgery comprising: a) administering a chlorotoxin conjugate to the individual wherein the chlorotoxin conjugate binds to residual cancer; and b) imaging, visualizing, or analyzing the bound chlorotoxin conjugate.
  • the chlorotoxin conjugate comprises one or more labeling agents.
  • the labeling agent comprises a fluorescent moiety.
  • the fluorescent moiety comprises a near infrared fluorescent moiety.
  • the labeling agent comprises a radionuclide.
  • the detecting is performed during or related to surgery or resection.
  • the residual cancer, or a portion thereof is removed during or related to surgery, surgical resection, or intraoperative imaging and resection.
  • the chlorotoxin conjugate comprises one or more labeling agents.
  • the labeling agent comprises a fluorescent moiety.
  • the fluorescent moiety comprises a near infrared fluorescent moiety.
  • the labeling agent comprises a radionuclide.
  • the detecting is performed during or related to surgery or resection.
  • the chlorotoxin conjugate is administered about 1 day before the surgery.
  • the chlorotoxin conjugate is administered about 2 days before surgery.
  • the chlorotoxin conjugate is administered in multiple sub-doses.
  • the chlorotoxin conjugate is administered in about 2 sub-doses, 3 sub-doses, 4 sub-doses, or more sub-doses.
  • the detecting comprises vivo, or ex vivo detection.
  • the imaging, visualizing, or analyzing comprises in vivo, or ex vivo imaging, visualizing, or analyzing.
  • a plot is made of the fluorescent intensity of the chlorotoxin conjugate.
  • the invention further provides methods for detecting soft tissue sarcoma in an individual comprising, administering a chlorotoxin conjugate to the individual, wherein the chlorotoxin conjugate comprises a detectable agent and a chlorotoxin polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR, binding the chlorotoxin conjugate to the soft tissue sarcoma, and detecting the bound chlorotoxin conjugate, wherein an elevated level of bound chlorotoxin conjugate indicates the presence of soft tissue sarcoma.
  • FIG. 1 shows SDS-PAGE of chlorotoxin conjugate formulations after 14 days at room temperature.
  • FIG. 2 shows SDS-PAGE of chlorotoxin conjugate formulations after 5 days at 40° C. (and t0).
  • FIG. 3 shows SDS-PAGE of chlorotoxin conjugate formulations after 3 days at room temperature in the light or 3 ⁇ F/T. Samples are after 3 days at room temperature in the light unless marked as F/T
  • FIG. 4 shows total peak area of RP-HPLC chromatograms for chlorotoxin conjugate formulations with no lyophilization or lyophilization with fast or slow freezing.
  • FIG. 5 shows FTIR-derived secondary structures obtained for chlorotoxin conjugate lyophilized with slow versus fast freezing (left) and in a liquid formulation (right).
  • FIG. 6 shows SDS-PAGE analysis of various lyophilized formulations.
  • FIG. 7 shows signal-to-noise ratios (SNRs) of chlorotoxin conjugates in U87 flank tumors 24 hours after injection.
  • FIG. 8 shows fluorescent images of Compound 76-2 kD (left), Compound 76-5 kD (middle) and Compound 76-40 kD (right).
  • FIG. 9A shows a fluorescent image of Compound 76-5 kD.
  • FIG. 9B shows an H&E stain of the tissue sample.
  • FIG. 10 shows biodistribution analysis of Compound 76-5 kD.
  • FIG. 10A shows integrated intensity for sections from liver, kidney, spleen, heart and brain.
  • FIGS. 10B and 10C show representative images from liver and kidney, respectively.
  • FIG. 11 shows an Odyssey fluorescent image of Compound 76-5 kD (top) and H&E stain (bottom) of heart tissue.
  • FIG. 12B shows signal from a subset of tumor and muscle samples. Representative muscle and tumor images for the 6 nmol and 20 nmol dose groups 1 and 3 days post injection are shown below the graph.
  • FIG. 13 shows biodistribution of select tissues using the IVIS Spectrum imaging system.
  • FIG. 13A represents tissues one day after injection.
  • FIG. 13B shows tissues imaged three days post injection. (B—brain, H—heart, K—kidney, S—skin, L—liver).
  • FIG. 13C shows fluorescent signal in tissues for the 2 nmol and 20 nmol groups one and three days after injection.
  • FIG. 14 shows whole body live animal imaging of chlorotoxin conjugate from six hours to three days after injection.
  • FIG. 15A shows ex vivo imaging of chlorotoxin conjugate 1 day after injection using the IVIS Spectrum.
  • FIG. 15B shows ex vivo imaging of the brain from orthotopic mouse 2.
  • FIG. 15C shows ex vivo Odyssey imaging of the brain and skull from orthotopic mouse 1.
  • FIG. 18 shows a determination of the chlorotoxin conjugate dose at which tumor to background ratio is maximal.
  • Sections of tumor and non-tumor tissue were scored by a histopathologist who was blinded to the fluorescence data.
  • Total fluorescence in 2 ⁇ 2 mm grid squares was measured, and each square was designated tumor or non-tumor by overlaying the fluorescence image with the H&E image scored by the histopathologist.
  • the average of tumor and non-tumor squares was used to compute the tumor to background ratio (TBR), shown at right of each data point.
  • TBR tumor to background ratio
  • FIG. 19 shows a box & whiskers plot of gross tumor intensity for soft-tissue sarcomas (all subtypes) and carcinomas (including adenocarcinoma and squamous cell carcinoma). Tissues were labeled with BLZ-100.
  • FIG. 20 shows signal and image analysis of tumor versus normal cortical tissue in SmoA1 mouse brain.
  • FIG. 21 shows small foci of fluorescence that correspond to small clusters of tumor cells highlighted in the H&E stained slide.
  • FIG. 22 shows the mean (SD) of scrum BLZ-100 concentration vs. time profiles following a single intravenous bolus of 0.02 mg BLZ-100 administered to female mice.
  • FIG. 23 shows the mean (SD) of serum BLZ-100 concentration vs. time profiles following a single intravenous bolus of 0.03 or 0.3 mg dose to male rats.
  • FIG. 24 shows the individual serum BLZ-100 concentration vs. time profiles following a single intravenous bolus dose of 0.6 mg to male monkeys.
  • FIG. 25 shows the mean ( ⁇ SD) BLZ-100 serum concentrations (ng/mL) summarized by dose following a single intravenous bolus dose to male and female rats.
  • FIG. 26 shows the mean ( ⁇ SD) BLZ-100 serum concentrations (ng/mL) summarized by dose following a single intravenous bolus dose to male and female monkeys.
  • FIG. 27 shows chlorotoxin conjugate serum concentration vs time for human subjects.
  • FIG. 28 shows intraoperative imaging of a soft-tissue sarcoma (patient 19).
  • A White light preoperative image of gross tumor showing ulcerated and grossly swollen peritumoral skin.
  • B NIR image of tumor in situ.
  • C Plot of fluorescence intensity along the line drawn through the image in panel B.
  • D NIR image of excised tumor.
  • E Plot of fluorescence intensity along the line drawn through the image in panel D.
  • F NIR image of peritumoral skin, surrounding uninvolved skin, and tumor bed. Peritumoral skin is 6-fold more intense than uninvolved skin. There is no residual fluorescence in the tumor bed. Tissues were labeled with BLZ-100.
  • FIG. 29 shows intraoperative imaging of a mammary carcinoma (patient 22).
  • A NIR image of fluorescence from the primary mass (arrow) imaged from the bottom of the resected tissue, taken immediately post-excision. This aspect had a margin of about 0.5 cm of normal tissue.
  • Panels B (fluorescence) and C (overlay) show the primary mass from the skin side, after the skin was opened and a slice removed for further imaging. The small fluorescent patches were originally part of the primary mass, but were separated by the removal of the slice.
  • Panels D and E show the gross appearance and fluorescence overlay of the pieces submitted for further imaging. The contrast between gross tumor and adjacent tissue is about 2.5-fold.
  • F Overlay image of the tumor bed, showing lack of residual fluorescence in the muscle wall. Tissues were labeled with BLZ-100.
  • FIG. 30 shows intraoperative imaging of a cutaneous squamous cell carcinoma (patient 23).
  • A White light preoperative image of tumor site, showing grossly ulcerated and swollen peritumoral skin.
  • Panels B and C show preoperative NIR fluorescence images of the tumor from the top (B) and side (C).
  • NIR fluorescence (D) and overlay (E) are shown following removal of the tail.
  • the mass at right is a section of central tumor removed for further analysis. The skin is retracted, and the remaining gross tumor (arrow) is revealed. Fluorescence intensity is similar in the central tumor and remaining gross tumor, while peritumoral skin has somewhat lower fluorescence intensity (F).
  • Tissues were labeled with BLZ-100.
  • FIG. 32 shows a box & whiskers plots of fluorescence intensity in grid squares for each tissue section analyzed.
  • T tumor.
  • NT adjacent non-tumor tissue.
  • PS peritumoral skin.
  • S uninvolved skin.
  • the NT consisted of underlying dermis, subcutaneous fat, and adjacent dermis/epidermis. Tissues were labeled with a chlorotoxin conjugate compound.
  • FIG. 33 shows imaging of a brain tumor labeled with BLZ-100.
  • A NIR fluorescence image of tumor in situ.
  • T tumor. M, nasal mucosa.
  • B normal brain.
  • Panels B and C show fluorescence images of 30 micron sections from two of the tumor pieces.
  • Panels D and E show H&E stains of the sections in B and C, respectively.
  • FIG. 16 shows fluorescence intensity in gross tumors, grouped by tumor type. Regions of interest (ROI) were drawn on Odyssey scans of gross tumors. ROIs were the same size across the data set.
  • ROI Regions of interest
  • FIG. 17 shows ex vivo imaging of a canine soft tissue sarcoma.
  • Patient 13 was a 7-year-old female Standard Poodle who presented with a subcutaneous hemangiopericytoma, a type of soft tissue sarcoma. She was treated with BLZ-100 at 0.94 mg/m 2 , and surgery was performed 48 hours later.
  • White light (A, C) and Odyssey near-infrared (B, D) images are shown of gross tumor and adjacent normal fat (A, B) and uninvolved skin (C, D). Gross ratios of tumor to fat were 257:1 to 127:1, and gross ratios of tumor to skin were 89:1 to 33:1.
  • FIG. 31 shows intraoperative imaging of a thyroid carcinoma (patient 20).
  • (Top) White light images of excised lymph node, thyroid mass, and tumor bed.
  • (Bottom) Monochrome NIR images. The lymph node and thyroid mass each have a section removed for further analysis, shown side-by-side with the bulk tissue.
  • compositions and methods for the detection and/or treatment of cancers comprise peptide conjugates comprising a detectable label, which are suitable for the detection and treatment of various cancers.
  • the compositions are provided in combination with a pharmaceutically acceptable carrier, which can be administered to a subject by any parenteral route of administration.
  • the compositions described herein give rise to a pharmacokinetic profile when administered intravenously to a human subject.
  • the conjugates bind selectively to cancer cells.
  • the cancer cells can then be detected, for example, by imaging or other visualization or detection method suitable for detecting the detectable label of the peptide conjugate.
  • the presently described compositions can be used to treat cancer by way of a therapeutic agent, which is attached to the conjugate and which acts on the cancer cells following binding by the peptide portion of the conjugate.
  • Niro refers to the —NO 2 radical.
  • Oxa refers to the —O— radical.
  • Oxo refers to the ⁇ O radical.
  • Thioxo refers to the ⁇ S radical.
  • “Hydrazino” refers to the ⁇ N—NH 2 radical.
  • Alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to fifteen carbon atoms (e.g., C 1 -C 15 alkyl). In certain embodiments, an alkyl comprises one to thirteen carbon atoms (e.g., C 1 -C 13 alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (e.g., C 1 -C 8 alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (e.g., C 5 -C 15 alkyl).
  • an alkyl comprises five to eight carbon atoms (e.g., C 5 -C 8 alkyl).
  • the alkyl is attached to the rest of the molecule by a single bond, for example, methyl (Me), ethyl (Et), n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like.
  • an alkyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, —OR a , —SR a , —OC(O)—R a , —N(R a ) 2 , —C(O)R a , —C(O)OR, —C(O)N(R a ) 2 , —N(R a )C(O)OR a , —N(R 2 )C(O)R a , —N(R a )S(O) t R a (where t is 1 or 2), —S(O) t OR a (where t is 1 or 2) and —S(O) t N(R a ) 2 (where t is 1 or 2) where each R a is independently hydrogen, alkyl, fluoroal
  • Alkenyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from two to twelve carbon atoms. In certain embodiments, an alkenyl comprises two to eight carbon atoms. In other embodiments, an alkenyl comprises two to four carbon atoms. The alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like.
  • an alkenyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, —OR a , —SR a , —OC(O)—R a , —N(R a ) 2 , —C(O)R a , —C(O)OR a , —C(O)N(R a ) 2 , —N(R a )C(O)OR a , —N(R a )C(O)R a , —N(R a )S(O) t R a (where t is 1 or 2), —S(O) t OR a (where t is 1 or 2) and —S(O) t N(R a ) 2 (where t is 1 or 2) where each R a is independently hydrogen, alkyl
  • Alkynyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having from two to twelve carbon atoms. In certain embodiments, an alkynyl comprises two to eight carbon atoms. In other embodiments, an alkynyl has two to four carbon atoms. The alkynyl is attached to the rest of the molecule by a single bond, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
  • an alkynyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, —OR, —SR a , —OC(O)—R a , —N(R a ) 2 , —C(O)R a , —C(O)OR a , —C(O)N(R a ) 2 , —N(R a )C(O)OR a , —N(R a )C(O)R a , —N(R a )S(O) t R a (where t is 1 or 2), —S(O) t OR a (where t is 1 or 2) and —S(O) t N(R a ) 2 (where t is 1 or 2) where each R a is independently hydrogen, alkyl, flu
  • Alkylene or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing no unsaturation and having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, n-butylene, and the like.
  • the alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond.
  • the points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon in the alkylene chain or through any two carbons within the chain.
  • an alkylene chain is optionally substituted by one or more of the following substituents: halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, thioxo, trimethylsilanyl, —OR a , —SR a , —OC(O)—R a , —N(R a ) 2 , —C(O)R a , —C(O)OR a , —C(O)N(R a ) 2 , —N(R a )C(O)OR a , —N(R a )C(O)R a , —N(R a )S(O) t R a (where t is 1 or 2), —S(O) t OR a (where t is 1 or 2) and —S(O) t N(R a ) 2 (
  • Alkenylene or “alkenylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing at least one double bond and having from two to twelve carbon atoms, for example, ethenylene, propenylene, n-butenylene, and the like.
  • the alkenylene chain is attached to the rest of the molecule through a double bond or a single bond and to the radical group through a double bond or a single bond.
  • the points of attachment of the alkenylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain.
  • an alkenylene chain is optionally substituted by one or more of the following substituents: halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, thioxo, trimethylsilanyl, —OR a , —SR a , —OC(O)—R a , —N(R a ) 2 , —C(O)R a , —C(O)OR a , —C(O)N(R a ) 2 , —N(R a )C(O)OR a , —N(R a )C(O)R a , —N(R a )S(O) t R a (where t is 1 or 2), —S(O) t OR a (where t is 1 or 2) and —S(O) t N(R a ) 2
  • Aryl refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon from six to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ⁇ -electron system in accordance with the Hückel theory.
  • Aryl groups include, but are not limited to, groups such as phenyl, fluorenyl, and naphthyl.
  • aryl or the prefix “ar-” (such as in “aralkyl”) is meant to include aryl radicals optionally substituted by one or more substituents independently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —R b —OR a , —R b —OC(O)—R a , —R b —N(R a ) 2 , —R b —C(O)R a ,
  • Alkyl refers to a radical of the formula —R c -aryl where R c is an alkylene chain as defined above, for example, benzyl, diphenylmethyl and the like.
  • the alkylene chain part of the aralkyl radical is optionally substituted as described above for an alkylene chain.
  • the aryl part of the aralkyl radical is optionally substituted as described above for an aryl group.
  • Alkenyl refers to a radical of the formula —R d -aryl where R d is an alkenylene chain as defined above.
  • the aryl part of the aralkenyl radical is optionally substituted as described above for an aryl group.
  • the alkenylene chain part of the aralkenyl radical is optionally substituted as defined above for an alkenylene group.
  • Alkynyl refers to a radical of the formula —R e -aryl, where R e is an alkynylene chain as defined above.
  • the aryl part of the aralkynyl radical is optionally substituted as described above for an aryl group.
  • the alkynylene chain part of the aralkynyl radical is optionally substituted as defined above for an alkynylene chain.
  • Carbocyclyl refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms. In certain embodiments, a carbocyclyl comprises three to ten carbon atoms. In other embodiments, a carbocyclyl comprises five to seven carbon atoms. The carbocyclyl is attached to the rest of the molecule by a single bond.
  • Carbocyclyl may be saturated, (i.e., containing single C—C bonds only) or unsaturated (i.e., containing one or more double bonds or triple bonds.)
  • a fully saturated carbocyclyl radical is also referred to as “cycloalkyl.”
  • monocyclic cycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • An unsaturated carbocyclyl is also referred to as “cycloalkenyl.”
  • Examples of monocyclic cycloalkenyls include, e.g., cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl.
  • Polycyclic carbocyclyl radicals include, for example, adamantyl, norbornyl (i.e., bicyclo[2.2.1]heptanyl), norbornenyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like.
  • carbocyclyl is meant to include carbocyclyl radicals that are optionally substituted by one or more substituents independently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —R b —OR a , —R b —SR a , —R b —OC(O)—R a , —R b —N(R a ) 2 , —R
  • Carbocyclylalkyl refers to a radical of the formula —R c -carbocyclyl where R c is an alkylene chain as defined above. The alkylene chain and the carbocyclyl radical is optionally substituted as defined above.
  • Halo or “halogen” refers to bromo, chloro, fluoro or iodo substituents.
  • Fluoroalkyl refers to an alkyl radical, as defined above, that is substituted by one or more fluoro radicals, as defined above, for example, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like.
  • the alkyl part of the fluoroalkyl radical is optionally substituted as defined above for an alkyl group.
  • Heterocyclyl refers to a stable 3- to 18-membered non-aromatic ring radical that comprises two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. The heteroatoms in the heterocyclyl radical may be optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocyclyl radical is partially or fully saturated. The heterocyclyl may be attached to the rest of the molecule through any atom of the ring(s).
  • heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thio
  • heterocyclyl is meant to include heterocyclyl radicals as defined above that are optionally substituted by one or more substituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —R b —OR a , —R b —SR a , —R b —OC(O)—R a , —R b —N(R a ) 2 , —
  • N-heterocyclyl or “N-attached heterocyclyl” refers to a heterocyclyl radical as defined above containing at least one nitrogen and where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a nitrogen atom in the heterocyclyl radical.
  • An N-heterocyclyl radical is optionally substituted as described above for heterocyclyl radicals. Examples of such N-heterocyclyl radicals include, but are not limited to, 1-morpholinyl, 1-piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl.
  • C-heterocyclyl or “C-attached heterocyclyl” refers to a heterocyclyl radical as defined above containing at least one heteroatom and where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a carbon atom in the heterocyclyl radical.
  • a C-heterocyclyl radical is optionally substituted as described above for heterocyclyl radicals. Examples of such C-heterocyclyl radicals include, but are not limited to, 2-morpholinyl, 2- or 3- or 4-piperidinyl, 2-piperazinyl, 2- or 3-pyrrolidinyl, and the like.
  • Heterocyclylalkyl refers to a radical of the formula —R c -heterocyclyl where R c is an alkylene chain as defined above. If the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl is optionally attached to the alkyl radical at the nitrogen atom.
  • the alkylene chain of the heterocyclylalkyl radical is optionally substituted as defined above for an alkylene chain.
  • the heterocyclyl part of the heterocyclylalkyl radical is optionally substituted as defined above for a heterocyclyl group.
  • Heteroaryl refers to a radical derived from a 3- to 18-membered aromatic ring radical that comprises two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur.
  • the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ⁇ -electron system in accordance with the Hückel theory.
  • Heteroaryl includes fused or bridged ring systems.
  • the heteroatom(s) in the heteroaryl radical is optionally oxidized.
  • heteroaryl is attached to the rest of the molecule through any atom of the ring(s).
  • heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothienyl (benzothion
  • heteroaryl is meant to include heteroaryl radicals as defined above which are optionally substituted by one or more substituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, haloalkenyl, haloalkynyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —R b —OR a , —R b —SR a , —R b —OC(O)—R a
  • N-heteroaryl refers to a heteroaryl radical as defined above containing at least one nitrogen and where the point of attachment of the heteroaryl radical to the rest of the molecule is through a nitrogen atom in the heteroaryl radical.
  • An N-heteroaryl radical is optionally substituted as described above for heteroaryl radicals.
  • C-heteroaryl refers to a heteroaryl radical as defined above and where the point of attachment of the heteroaryl radical to the rest of the molecule is through a carbon atom in the heteroaryl radical.
  • a C-heteroaryl radical is optionally substituted as described above for heteroaryl radicals.
  • Heteroarylalkyl refers to a radical of the formula —R c -heteroaryl, where R c is an alkylene chain as defined above. If the heteroaryl is a nitrogen-containing heteroaryl, the heteroaryl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain of the heteroarylalkyl radical is optionally substituted as defined above for an alkylene chain. The heteroaryl part of the heteroarylalkyl radical is optionally substituted as defined above for a heteroaryl group.
  • the compounds, or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids.
  • the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E (or trans) and Z (cis) geometric isomers.
  • all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended to be included.
  • stereoisomer refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. It is therefore contemplated that various stereoisomers and mixtures thereof and includes “enantiomers,” which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another.
  • a “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule.
  • the compounds presented herein may exist as tautomers.
  • Tautomers are compounds that are interconvertible by migration of a hydrogen atom, accompanied by a switch of a single bond and adjacent double bond. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will exist. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH.
  • “Pharmaceutically acceptable salt” includes both acid and base addition salts.
  • a pharmaceutically acceptable salt of any one of the alkoxyphenyl-linked amine derivative compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms.
  • Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and, aromatic sulfonic acids, etc.
  • acetic acid trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like.
  • salts of amino acids such as arginates, gluconates, and galacturonates
  • Acid addition salts of basic compounds may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.
  • “Pharmaceutically acceptable base addition salt” refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. See Berge et al
  • treatment or “treating,” or “palliating” or “ameliorating” are used interchangeably herein. These terms refers to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated.
  • a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.
  • the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
  • Prodrug is meant to indicate a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound described herein.
  • prodrug refers to a precursor of a biologically active compound that is pharmaceutically acceptable.
  • a prodrug may be inactive when administered to a subject, but is converted in vivo to an active compound, for example, by hydrolysis.
  • the prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam).
  • prodrugs are provided in Higuchi, T., et al., “Pro-drugs as Novel Delivery Systems,” A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated in full by reference herein.
  • prodrug is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a mammalian subject.
  • Prodrugs of an active compound, as described herein may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent active compound.
  • Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a mammalian subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively.
  • Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol or amine functional groups in the active compounds and the like.
  • the present disclosure provides compounds that selectively bind to cancerous cells and tissues.
  • the compounds of the present disclosure comprise a peptide portion and a detectable agent conjugated together.
  • the peptide portions of the compounds described herein have certain features in common with the native chlorotoxin (CTX) peptide.
  • Chlorotoxin is a 36 amino acid peptide that selectively binds to cancerous cells.
  • the peptide portions of the present compounds have advantageously retained at least some of the cancer-cell binding activity of chlorotoxin.
  • the cancer-cell binding activity of chlorotoxin provides certain advantages for the detection and treatment of cancer because it facilitates the selective localization of detectable agents and therapeutic agents to the cancer cells for the detection and treatment of cancer.
  • Citrulline is designated as “Cit” in the sequences.
  • Chlorotoxin conjugates comprise a chlorotoxin and a labeling agent or detectable label.
  • chlorotoxin is a variant comprising at least 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of the natural peptide of chlorotoxin.
  • the present disclosure provides a chlorotoxin having the following amino acid sequence: MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR.
  • the present disclosure provides chlorotoxin variants comprising at least 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following amino acid sequence: MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR.
  • the chlorotoxin is a chlorotoxin or variant thereof comprising at least 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR, wherein X is selected from K, A and R.
  • the chlorotoxin is a chlorotoxin or variant of thereof comprising at least 85% sequence identity to the sequence of MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR, wherein X is selected from K, A and R.
  • the chlorotoxin is BLZ-100, which is a chlorotoxin variant comprising the sequence of MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR, wherein X15 and X23 are arginine and X27 is lysine conjugated to a cyanine fluorescent label.
  • the peptide canbe further cross-linked by four disulfide bonds formed among the cysteine residues present in the sequence.
  • the peptide is a variant of the natural peptide of chlorotoxin but retains all eight cysteine residues of the natural peptide, enabling cross-linking by up to four disulfide bonds. Conservation of cysteine residues helps to preserve the secondary structure, charge distribution, isolelectric point (pI) and other features of the natural chlorotoxin peptide because of the disulfide bonds that form between the cysteine residues.
  • the chlorotoxin peptide variant retains all eight cysteine residues of the natural peptide and has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the native chlorotoxin peptide.
  • the chlorotoxin peptide variant has eight cysteine residues positioned so that the distances between pairs of cysteines is the same as the distances between pairs of cysteines found in the natural peptide, and the chlorotoxin peptide variant has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the native chlorotoxin peptide.
  • the chlorotoxin peptide variant has eight cysteine residues positioned so that the distances between pairs of cysteines is functionally equivalent or functionally similar to the distances between pairs of cysteines found in the natural peptide, and the chlorotoxin peptide variant has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the native chlorotoxin peptide.
  • the chlorotoxin peptide variant has eight cysteine residues positioned so that the distances between pairs of cysteines allows for secondary structure and isolectric point of the native chlorotoxin peptide to be preserved, and the chlorotoxin peptide variant has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the native chlorotoxin peptide.
  • the chlorotoxin peptide variant has eight cysteine residues positioned so that the distances between pairs of cysteines is sufficient to allow disulfide bonds to form, and the chlorotoxin peptide variant has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the native chlorotoxin peptide.
  • one or more methionines of the chlorotoxin peptide variant are replaced with other amino acids. In some aspects, one or more methionines of the chlorotoxin peptide variant are replaced with other amino acids selected from glycine, alanine, Isoleucine, Threonine, Valine, Leucine, Serine or a combination thereof.
  • the chlorotoxin can be a chlorotoxin variant.
  • Chlorotoxin and chlorotoxin variants have are further described in PCT Patent Application Publication Numbers WO2006115633 and WO2011142858, which are incorporated in their entirety herein by reference.
  • the peptide can have the following formula: H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Xaa-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Xaa-Gly-Arg-Gly-Xaa-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH acetate salt (disulfide bonds, air oxidized), wherein Xaa is Arg, Ala, or Lys.
  • the all peptide can have the following formula: H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Xaa-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Xaa-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg- OH acetate salt (disulfide bonds, air oxidized), wherein Xaa is Arg, or Ala.
  • the peptide can have the following formula: H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Arg-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Arg-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH acetate salt (disulfide bonds, air oxidized).
  • the peptide can have the following formula: H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Arg-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Ala-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH acetate salt (disulfide bonds, air oxidized).
  • the peptide can have the following formula: H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Ala-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Arg-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH acetate salt (disulfide bonds, air oxidized).
  • the peptide can have the following formula: H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Ala-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Ala-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH acetate salt (disulfide bonds, air oxidized).
  • the chlorotoxin and chlorotoxin variants can be conjugated to moieties, such as detectable labels (e.g., dyes) that can be detected (e.g., visualized) in a subject.
  • detectable labels e.g., dyes
  • the chlorotoxin and/or chlorotoxin variants can be conjugated to detectable labels to enable tracking of the bio-distribution of a conjugated peptide.
  • the detectable labels can include fluorescent dyes.
  • Non-limiting examples of fluorescent dyes that could be used as a conjugating molecule in the present disclosure include rhodamine, rhodol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol, methylrhodol, sulforhodol; aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, and thiorhodamine, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, a cyanine dye (e.g., cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5,
  • Some other example dyes include near-infrared dyes, such as, but not limited to, Cy5.5, indocyanine green (ICG), DyLight 750 or IRdye 800.
  • near infrared dyes can include cyanine dyes.
  • Chemotherapueutics, anti-cancer drugs, and anti-cancer agents include, but are not limited to: radioisotopes, toxins, enzymes, sensitizing drugs, nucleic acids, including interfering RNAs, antibodies, anti-angiogenic agents, cisplatin, anti-metabolites, mitotic inhibitors, growth factor inhibitors, paclitaxel, temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, decarbazine, altretamine, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, mitomycin, bleomycin
  • the terms “about” and “approximately,” in reference to a number, is used herein to include numbers that fall within a range of 10%, 5%, or 1% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • Suitable diagnostic agents include agents that provide for the detection by fluorescence methods as well as methods other than fluorescence imaging.
  • Other suitable diagnostic agents include radiolabels (e.g., radio isotopically labeled compounds) such as 125 I, 14 C, and 31 P, among others; and magnetic resonance imaging agents.
  • Suitable targeting agents include antibodies, polypeptides, polysaccharides, and nucleic acids.
  • compositions that include the modified chlorotoxin peptide conjugates are provided.
  • the composition can include a pharmaceutically acceptable carrier or diluent for delivery of the modified chlorotoxin peptide conjugate.
  • Suitable pharmaceutically acceptable carriers or diluents include saline or dextrose for injection.
  • the presently described compounds further comprise a detectable label, which can be used for the detection of the peptide-label conjugate and the cancerous cells to which they are bound.
  • compounds of the present disclosure have the structure of Formula (I), or a pharmaceutically acceptable salt thereof:
  • the presently described compounds further comprise a detectable label, which can be used for the detection of the peptide-label conjugate and the cancerous cells to which they are bound.
  • compounds of the present disclosure have the structure of Formula (XV), or a pharmaceutically acceptable salt thereof:
  • the compounds of the present disclosure have a structure of Formula (II), or a pharmaceutically acceptable salt thereof:
  • the present compounds have a structure of Formula (III), or a pharmaceutically acceptable salt thereof:
  • compounds of the present disclosure have a structure of Formula (IV), or a pharmaceutically acceptable salt thereof:
  • compounds of the present disclosure have a structure of Formula (V), or a pharmaceutically acceptable salt thereof:
  • compounds of the present disclosure have a structure of Formula (VI), or a pharmaceutically acceptable salt thereof:
  • compounds of the present disclosure have a structure Formula (III), or a pharmaceutically acceptable salt thereof:
  • a 1 , A 2 , and A 3 are absent.
  • a 5 is hydrogen.
  • R 3 , R 4 , R 5 , and R 6 are each independently C 1 -C 6 alkyl.
  • R 3 , R 4 , R 5 , R 6 are each independently methyl.
  • R 1 , R 2 , R 7 , R 8 , R 15 , and R 16 are each independently selected from hydrogen or sulfonate.
  • R 1 , R 2 , R 7 , R 8 , R 15 , and R 16 are each independently hydrogen.
  • R 12 , R 13 , R 14 , R 19 , R 20 are each independently hydrogen.
  • R 12 and R 13 join together along with the atoms to which they are attached to form a six-membered carbocyclic ring. In other aspects, R 12 and R 13 join together along with the atoms to which they are attached to form a five-membered carbocyclic ring. In certain aspects, R 14 and R 19 join together along with the atoms to which they are attached to form a six-membered carbocyclic ring. In some aspects, R 14 and R 20 join together along with the atoms to which they are attached to form a six-membered carbocyclic ring. In certain aspects, L 1 is C 3 -C 6 alkylene. In other aspects, L 1 is C 3 -C 5 alkylene.
  • L 1 is propylene. In still other aspects, L L is butylene. In other aspects, L 1 is pentylene. In some aspects, L 2 is C 3 -C 6 alkylene. In other aspects, L 2 is propylene. In still other aspects, L 2 is butylene. In other aspects, L 2 is pentylene. In some aspects, R 9 is sulfonate. In other aspects, R 9 is hydrogen. In some aspects, R 14 is hydrogen. In other aspects, R 14 is -(L 5 )-aryl. In still other aspects, R 14 is (L 5 )-aryl-A 5 .
  • R 1 is hydrogen. In certain aspects, R 2 is hydrogen. In some aspects, R 3 is methyl. In certain aspects, R 4 is methyl. In some aspects, R 5 is methyl. In certain aspects R 6 is methyl. In some aspects, R 7 is hydrogen. In certain aspects, R 8 is hydrogen. In some aspects, R 12 is hydrogen. In certain aspects, R 13 is hydrogen. In some aspects, R 14 is hydrogen. In certain aspects, R 19 is hydrogen. In some aspects, R 20 is hydrogen. In certain aspects, R 10 is hydrogen. In some aspects, R 11 is hydrogen.
  • R 17 and R 18 are independently phenyl.
  • L 3 is selected from a bond, —O—, —NR 10 —, —NR 10 —C 1 -C 6 alkylene-, —O—NR 10 —, or —NR 10 -L 4 -. In further aspects, L 3 is a bond.
  • L 4 is -heterocyclyl- or -heterocyclyl-C 1 -C 6 alkylene-. In further aspects, L 4 is -piperizinyl-(C 1 -C 6 alkylene). In still further aspects, L 4 is
  • p is 1. In certain aspects, q is 1.
  • the compound has the structure of any one of Formulas (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV):
  • one of A 1 , A 2 , A 3 , A 4 , or A 5 is a polypeptide having at least 87% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.
  • one of A 1 , A 2 , A 3 , A 4 , or A 5 is a polypeptide having at least 90% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.
  • one of A 1 , A 2 , A 3 , A 4 , or A 5 is a polypeptide having at least 92% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.
  • one of A 1 , A 2 , A 3 , A 4 , or A 5 is a polypeptide having at least 95% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. In still further aspects, one of A 1 , A 2 , A 3 , A 4 , or A 5 is a polypeptide having at least 97% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. In still further aspects, one of A 1 , A 2 , A 3 , A 4 , or A 5 is a polypeptide having 100% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. In still further aspects, one of A 1 , A 2 , A 3 , A 4 , or A 5 is a polypeptide having the sequence MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.
  • the fragment of A 1 , A 2 , A 3 , A 4 , or A 5 has a length of at least 25 amino acid residues. In further aspects, the fragment of A 1 , A 2 , A 3 , A 4 , or A 5 has a length of at least 27 amino acid residues. In still further aspects, the fragment of A 1 , A 2 , A 3 , A 4 , or A 5 has a length of at least 29 amino acid residues. In still further aspects, the fragment of A 1 , A 2 , A 3 , A 4 , or A 5 has a length of at least 31 amino acid residues. In still further aspects, the fragment of A 1 , A 2 , A 3 , A 4 , or A 5 has a length of at least 33 amino acid residues.
  • one of A 1 , A 2 , A 3 , A 4 , or A 5 is a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof having the tumor cell binding affinity of native chlorotoxin. In certain aspects, one of A 1 , A 2 , A 3 , A 4 , or A 5 is a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof having essentially the same the tumor cell binding affinity of native chlorotoxin.
  • one of A 1 , A 2 , A 3 , A 4 , or A 5 is a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof having the tumor cell binding affinity of native chlorotoxin wherein one of A 1 , A 2 , A 3 , A 4 , or A 5 has a sequence selected from SEQ ID NOS: 1-481.
  • the polypeptide comprises at least one lysine amino acid residue. In certain aspects, the polypeptide comprises a single lysine amino acid residue. In some aspects, the polypeptide comprises one, two, or three lysine amino acid residues. In some aspects, the polypeptide comprises a lysine residue at the position corresponding to K-27 of native chlorotoxin. In some aspects, the polypeptide comprises a lysine residue at the position corresponding to K-23 of native chlorotoxin. In some aspects, the polypeptide comprises a lysine residue at the position corresponding to K-15 of native chlorotoxin.
  • one or more of the amino acids of the polypeptide is substituted with a non-naturally occurring amino acid residue.
  • the non-naturally occurring amino acid residue is a citrulline amino acid residue.
  • L 3 is attached to A 4 at a citrulline amino acid residue of the polypeptide.
  • L 3 is attached to A 4 at a lysine amino acid residue of the polypeptide. In certain aspects, L 3 is attached to A 4 at the N-terminus of the polypeptide. In some aspects, L 3 is attached to A 4 at the C-terminus of the polypeptide. In some aspects, the R 3 is attached to A 1 at a lysine amino acid residue of the peptide, a citrulline amino acid residue of the polypeptide, the N-terminus of the polypeptide, or the C-terminus of the polypeptide.
  • the R 5 is attached to A 2 at a lysine amino acid residue of the polypeptide, a citrulline amino acid residue of the polypeptide, the N-terminus of the polypeptide, or the C-terminus of the polypeptide.
  • the R 9 is attached to A 3 at a lysine amino acid residue of the polypeptide, a citrulline amino acid residue of the polypeptide, the N-terminus of the polypeptide, or the C-terminus of the polypeptide.
  • the aryl is attached to A 5 at a lysine amino acid residue of the polypeptide, a citrulline amino acid residue of the polypeptide, the N-terminus of the polypeptide, or the C-terminus of the polypeptide.
  • the compound has the structure of any one of compounds 1 to 721 as found in Tables 2-13.
  • the compound is conjugated to polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), an albumin derivative, or a fatty acid.
  • PEG polyethylene glycol
  • hydroxyethyl starch polyvinyl alcohol
  • a water soluble polymer a zwitterionic water soluble polymer
  • a water soluble poly(amino acid) an albumin derivative
  • a fatty acid or a fatty acid
  • the polypeptide has an isoelectric point of from 7.5 to 9.0. In some aspects, the polypeptide has an isoelectric point of from 8.0 to 9.0. In some aspects, the polypeptide has an isoelectric point of from 8.5 to 9.0. In some aspects, the polypeptide is basic and has an isoelectric point of greater than 7.5.
  • the polypeptide comprises at least eight cysteine amino acid residues. In some aspects, the polypeptide comprises eight cysteine amino acid residues. In some aspects, the polypeptide comprises four disulfide bonds. In some aspects, the polypeptide comprises from six to seven cysteine amino acid residues. In some aspects, the polypeptide comprises three disulfide bonds. In some aspects, the spacing between the cysteine amino acid residues in the polypeptide is essentially the same as in native chlorotoxin. In some aspects, the distribution of charge on the surface of the polypeptide is essentially the same as in native chlorotoxin.
  • one or more of the methionine amino acid residues is replaced with an amino acid residue selected from isoleucine, threonine, valine, leucine, serine, glycine, alanine, or a combination thereof.
  • the compound is capable of passing across the blood brain barrier.
  • the compound further comprises a therapeutic agent attached to A.
  • the therapeutic agent is a cytotoxic agent.
  • the present disclosure provides a composition
  • a composition comprising a compound comprising a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof, wherein when the composition is intravenously administering to a human subject at a dose of from 1 mg to 30 mg, the composition produces in the human subject an average maximum compound blood plasma concentration (average C max ) of at least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of the compound administered.
  • average C max average maximum compound blood plasma concentration
  • the compound of the composition is any suitable compound described in the present disclosure.
  • A MCMPCFTTDHQMARACDDCCGGAGRG K CYGPQCLCR (SEQ ID NO: 5) (attached at K-27) No. Structure 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
  • A MCMPCFTTDHQMARRCDDCCGGAGRG K CYGPQCLCR (SEQ ID NO: 6) (attached at K-27) No. Structure 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180
  • A MCMPCFTTDHQMARACDDCCGGRGRG K CYGPQCLCR (SEQ ID NO: 8) (attached at K-27) No. Structure 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240
  • A MCMPCFTTDHQMAR K CDDCCGGRGRGACYGPQCLCR (SEQ ID NO: 16) (attached at K-15) No. Structure 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470
  • A MCMPCFTTDHQMARACDDCCGG K GRGRCYGPQCLCR (SEQ ID NO: 20) (attached at K-23) No. Structure 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530
  • A MCMPCFTTDHQMARRCDDCCGG K GRGRCYGPQCL (SEQ ID NO: 21) (attached at K-23) No. Structure 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590
  • the presently described peptides are conjugated to moieties, such as detectable labels (e.g., dyes or radiolabels) that are detected (e.g., visualized) in a subject.
  • detectable labels e.g., dyes or radiolabels
  • the chlorotoxin and/or chlorotoxin variants is conjugated to detectable labels to enable tracking of the bio-distribution of a conjugated peptide.
  • the fluorescent moiety is covalently coupled to the chlorotoxin to allow for the visualization of the conjugate by fluorescence imaging, either directly or through a linker as described herein and known to one of ordinary skill in the art.
  • the fluorescent label has emission characteristics that are desired for a particular application.
  • the fluorescent label is a fluorescent dye that has a emission wavelength maximum between a range of 500 nm to 1100 nm, between a range of 600 nm to 1000 nm, between a range of 600 to 800 nm, between a range of 650 nm to 850 nm, or between a range of 700 nm to 800 nm.
  • the fluorescent label is a fluorescent dye that has a emission wavelength maximum between a range of about 500 nm to about 1100 nm, between a range of about 600 nm to about 1000 nm, between a range of about 600 to about 800 nm, between a range of about 650 nm to about 850 nm, or between a range of about 700 nm to about 800 nm.
  • a fluorescent dye that has a emission wavelength maximum between a range of about 500 nm to about 1100 nm, between a range of about 600 nm to about 1000 nm, between a range of about 600 to about 800 nm, between a range of about 650 nm to about 850 nm, or between a range of about 700 nm to about 800 nm.
  • near-infrared dyes such as, but not limited to, DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, or indocyanine green (ICG).
  • near infrared dyes often include cyanine dyes.
  • Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.), cou
  • the conjugate compounds include a chemiluminescent compound, colloidal metal, luminescent compound, enzyme, radioisotope, or paramagnetic labels.
  • the conjugates of the present disclosure are conjugated to radioactive isotopes instead of or in addition to other types of detectable agents.
  • Certain isotopes sutable for use in the present compounds include, but not limited to, iodine-131, iodine-125, bismuth-212, bismuth-213, lutetium-177, rhenium-186, rhenium-188, yttrium-90, astatine-211, phosphorus-32 and/or samarium-153.
  • the conjugates of the present disclosure contain one or more atoms having an atomic mass or mass number different from the atomic mass or mass number usually found in nature, including but not limited to hydrogen, carbon, fluorine, phosphorous, copper, gallium, yttrium, technetium, indium, iodine, rhenium, thallium, bismuth, astatine, samarium, and lutetium (for example, 3 H, 3 H, 13 C, 14 C, 18 F, 32 P, 35 S, 64 Cu, 67 Ga, 90 Y, 99M Tc, 111 In, 125 I, 123 I, 131 I, 135 I, 186 Re, 187 Re.
  • hydrogen, carbon, fluorine, phosphorous, copper, gallium, yttrium, technetium, indium, iodine, rhenium, thallium, bismuth, astatine, samarium, and lutetium for example, 3 H, 3 H, 13 C, 14 C
  • the conjugates of the present disclosure are labeled with a paramagnetic metal ion that is a good contrast enhancer in Magnetic Resonance Imaging (MRI).
  • MRI Magnetic Resonance Imaging
  • paramagnetic metal ions include, but are not limited to, gadolinium III (Gd3+), chromium 111 (Cr3+), dysprosium III (Dy3+), iron 111 (Fe3+), manganese II (Mn2+), and ytterbium III (Yb3+).
  • the labeling moieties comprises gadolinium III (Gd3+)
  • the conjugates of the present disclosure are conjugated to biotin.
  • biotin also acts as an affinity handle for retrieval of the peptides from tissues or other locations.
  • the conjugates are conjugated, e.g., to a biotinidase resistant biotin with a PEG linker (e.g., NHS-dPEG4-Biotinidase resistant biotin).
  • fluorescent biotin conjugates that can act both as a detectable label and an affinity handle are used.
  • Non-limiting examples of commercially available fluorescent biotin conjugates include Atto 425-Biotin, Atto 488-Biotin, Atto 520-Biotin, Atto-550 Biotin, Atto 565-Biotin, Atto 590-Biotin, Atto 610-Biotin, Atto 620-Biotin, Atto 655-Biotin, Atto 680-Biotin, Atto 700-Biotin, Atto 725-Biotin, Atto 740-Biotin, fluorescein biotin, biotin-4-fluorescein, biotin-(5-fluorescein) conjugate, and biotin-B-phycoerythrin, alexa fluor 488 biocytin, alexa flour 546, alexa fluor 549, lucifer yellow cadaverine biotin-X, Lucifer yellow biocytin, Oregon green 488 biocytin, biotin-rhodamine and tetramethylr
  • the peptides of the present disclosure are directly conjugated to a detectable label, such as a dye, fluorescent moiety or the like such that no additional amino acids, carbohydrates, nucleic acids, polymers, organic chains, or the like are added to the chlorotoxin or chlorotoxin variant and/or the dye, fluorescent moiety or the like to comprise the chlorotoxin conjugates described herein.
  • a detectable label such as a dye, fluorescent moiety or the like such that no additional amino acids, carbohydrates, nucleic acids, polymers, organic chains, or the like are added to the chlorotoxin or chlorotoxin variant and/or the dye, fluorescent moiety or the like to comprise the chlorotoxin conjugates described herein.
  • a linker is used to conjugate the chlorotoxin or chlorotoxin variant is not directly conjugated to a dye, fluorescent moiety or the like such that additional amino acids, carbohydrates, nucleic acids or the like are added to the chlorotoxin or chlorotoxin variant and/or the dye, fluorescent moiety or the like to comprise the chlorotoxin conjugates described herein.
  • a “linker” as used herein refers to at least one compound comprising two functional groups that are capable of reacting specifically with other moieties to form covalent or non-covalent linkages. Such moieties include, but are not limited to, the side groups on natural or non-natural amino acids or peptides which contain such natural or non-natural amino acids.
  • a linker has a functional group reactive with a group on a first peptide, and another functional group which is reactive with a group on a second peptide, whereby forming a conjugate that includes the first peptide, the linker and the second peptide.
  • Many procedures and linker molecules for attachment of various compounds to peptides are known. See, e.g., European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789 which are incorporated by reference herein in their entirety.
  • linkage refers to a bond or a chemical moiety formed from a chemical reaction between the functional group of a linker and another molecule.
  • bonds include, but are not limited to, covalent linkages and non-covalent bonds, while such chemical moieties include, but are not limited to, esters, carbonates, imines phosphate esters, hydrazones, acetals, orthoesters, peptide linkages, and oligonucleotide linkages.
  • Hydrolytically stable linkages means that the linkages are substantially stable in water and do not react with water at neutral pH values, including but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely.
  • Hydrolytically unstable or degradable linkages mean that the linkages are degradable in water or in aqueous solutions, including for example, blood.
  • Enzymatically unstable or degradable linkages mean that the linkage is often degraded by one or more enzymes.
  • PEG and related polymers include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule.
  • Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent, wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent.
  • hydrolytically degradable linkages include but are not limited to carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5′ hydroxyl group of an oligonucleotide.
  • the conjugates for use in the method described herein is conjugated by using any art-recognized method forming a complex including covalent, ionic, or hydrogen bonding of the ligand to the imaging agent, either directly or indirectly via a linking group such as a linker.
  • the conjugate is typically formed by covalent bonding of the ligand to the imaging agent through the formation of amide, ester or imino bonds between acid, aldehyde, hydroxy, amino, or hydrazo groups on the respective components of the complex or, for example, by the formation of disulfide bonds.
  • linker portion of the conjugates are contemplated herein.
  • a number of amino acid substitutions are often made to the linker portion of the conjugate, including but not limited to naturally occurring amino acids, as well as those available from conventional synthetic methods.
  • beta, gamma, and longer chain amino acids are used in place of one or more alpha amino acids.
  • the stereochemistry of the chiral centers found in such molecules is selected to form various mixture of optical purity of the entire molecule, or only of a subset of the chiral centers present.
  • the length of the peptide chain included in the linker is shortened or lengthened, either by changing the number of amino acids included therein, or by including more or fewer beta, gamma, or longer chain amino acids.
  • the selection of amino acid side chains in the peptide portion is made to increase or decrease the relative hydrophilicity of the linker portion specifically or of the overall molecule generally.
  • the linker includes an alkylene chain.
  • the alkylene chain often varies in length, or includes branched groups, or includes a cyclic portion, which are in line or spiro relative to the allylene chain.
  • linker includes a beta thiol releasable fragment
  • other intervening groups connecting the thiol end to the hydroxy or carbonate end are used in place of the ethylene bridge, such as but not limited to optionally substituted benzyl groups, where the hydroxy end is connected at the benzyl carbon and the thiol end is connected through the ortho or para phenyl position, and vice versa.
  • compositions comprising the above-described compounds and a pharmaceutically acceptable carrier.
  • the composition is formulated for parenteral administration.
  • the composition is formulated for intravenous administration, intramuscular administration, subcutaneous administration, or a combination thereof.
  • Intravenous pharmaceutical compositions of chlorotoxin conjugates include any formulation suitable for administration to a subject via any intravenous method, including a bolus, an infusion which occurs over time or any other intravenous method known in the art.
  • the rate of infusion is such that the dose is administered over a period of less than five minutes, more than five minutes but less than 15 minutes or greater than 15 minutes.
  • the rate of infusion is such that the dose is administered over a period of less than 5 minutes.
  • the rate of infusion is such that the dose is administered over a period of greater than 5 minutes and less than 15 minutes.
  • the rate of infusion is such that the dose is administered over a period of greater than 15 minutes.
  • “Product” or “dosage form” as used herein refers to any solid, semi-solid, lyophilized, aqueous, liquid or frozen formulation or preparation used for administration.
  • the rate of release of an active moiety from a product is often greatly influenced by the excipients and/or product characteristics which make up the product itself.
  • an enteric coat on a tablet is designed to separate that tablet's contents from the stomach contents to prevent, for example, degradation of the stomach which often induces gastrointestinal discomfort or injury.
  • systemic exposure of the active moiety will be relatively insensitive to the small formulation changes.
  • “pharmaceutically acceptable” or “pharmacologically acceptable” includes molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a subject, as appropriate.
  • “Pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients are often also incorporated into the compositions.
  • the present compositions comprise a concentration of the compound as an active pharmaceutical ingredient having a concentration of from 1 mg/mL to 40 mg/mL. In further aspects, the concentration of the compound is from 1 mg/mL to 20 mg/mL. In still other aspects, the concentration of the compound is from 4 mg/mL to 10 mg/mL. In additional aspects, the concentration of the compound is from 5 mg/mL to 8 mg/mL. In yet further aspects, concentration of the compound is from 5 mg/mL to 6 mg/mL.
  • pharmaceutically acceptable carrier comprises tris, D-mannitol, and a pH of essentially 6.8.
  • the compositions consist essentially of tris, D-mannitol, and a pH of 6.8.
  • pharmaceutically acceptable carrier comprises histidine and mannitol. In some aspects, pharmaceutically acceptable carrier comprises histidine and mannitol with polysorbate 20. In some aspects, pharmaceutically acceptable carrier comprises L-histidine, D-mannitol, L-methionine, and a pH of essentially 6.8. In additional aspects, the pharmaceutically acceptable carrier consists essentially of L-histidine, D-mannitol, L-methionine, and a pH of 6.8.
  • the pharmaceutically acceptable carrier comprises L-histidine, D-mannitol, polysorbate 20, and a pH of essentially 6.8. In some aspects, the pharmaceutically acceptable carrier comprises L-histidine, D-mannitol, and a pH of essentially 6.8. In some aspects, the pharmaceutically acceptable carrier comprises L-histidine, D-mannitol, polysorbate 20, trehalose, and a pH of essentially 6.8.
  • a pharmaceutical composition comprising a chlorotoxin conjugate is formulated according to known methods to prepare pharmaceutically useful compositions, for example, as found in “Excipient Selection in Parenteral Formulation Development” Pramanick et. al., Pharma Times, Vol. 45, No. 3, March 2013, incorporated in its entirety herein by reference.
  • the chlorotoxin conjugate is combined with a pharmaceutically acceptable carrier.
  • a composition is said to be a pharmaceutically acceptable carrier if its administration is tolerated by a recipient patient.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • Other suitable carriers are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • Formulations for administration of chlorotoxin conjugates are typically provided but are not limited to as liquid, solid or semi-solid products or dosage forms, exemplified by tablets, capsules, pellets, a powder or a lyophilized product.
  • the chlorotoxin conjugate is formulated to comprise no additional materials except for a pharmaceutical carrier.
  • the chlorotoxin conjugate is formulated such that it comprises a core “matrix material” which encapsulates, binds to, coats or is adjacent to the chlorotoxin conjugate.
  • the chlorotoxin conjugate and matrix material further comprises a protective coatings.
  • Various formulations are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • Suitable excipients for use with chlorotoxin conjugates are often included in formulations for intravenous use, for example, an injection.
  • Injections are sterile, pyrogen-free solutions or dispersions (emulsions or suspensions) of one or more active ingredients in a suitable vehicle or carrier.
  • Injections that are dispersions should remain sufficiently stable so that, after shaking, a homogeneous dose is withdrawn.
  • formulations which include chlorotoxin conjugates and one or more but not limited to suitable excipients, exemplified by matrix materials, binders, lubricants, glidants or disintegrants which aid in modulating the PK profile of administered chlorotoxin conjugates are preferred.
  • compositions comprising chlorotoxin conjugates in combination with one or more suitable excipients and one or more specific product characteristics (such as dissolution or water content) which result in improved pharmacokinetic profiles of chlorotoxin conjugates in vivo.
  • specific product characteristics such as dissolution or water content
  • the in vivo performance of chlorotoxin conjugates dosage forms/products included herein is based upon the composition of the excipients added during manufacturing and/or the final product characteristics generated through specific processing parameters and methods.
  • Other excipients are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • Suitable carriers for intravenous administration include for example but are not limited to physiological saline or phosphate buffered saline (PBS), Tris, and solutions containing solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol, additional agents such as histidine, dextrose, mannitol and mixtures thereof.
  • carriers for intravenous administration include a mixture of histidine and dextrose, Tris and dextrose or Tris and mannitol.
  • Other carriers are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • the formulation often includes an aqueous vehicle.
  • Aqueous vehicles include, by way of example and without limitation, sodium chloride solution, Ringers solution, isotonic dextrose solution, sterile water solution, dextrose and lactated Ringers solution.
  • Nonaqueous vehicles include, by way of example and without limitation, fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil, benzyl benzoate, castor oil, N,N-dimethylacetamide, ethanol, dehydrated ethanol, glycerin, glycerol, N-methyl-2-pyrrolidone, polyethylene glycol and any derivative thereof, propylene glycol, safflower oil and soybean oil.
  • Other vehicles are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • the composition the pharmaceutically acceptable carrier comprises an osmolyte.
  • the osmolyte comprises a sugar, a sugar alcohol, or a combination thereof.
  • the composition comprises a sugar alcohol selected from sorbitol, inositol, mannitol, xylitol and glycerol, or a combination thereof.
  • the sugar alcohol comprises mannitol.
  • the composition comprises from 2% to 20% (wt/vol %) mannitol.
  • the composition comprises from 2% to 10% (wt/vol %) mannitol.
  • the composition comprises essentially 5% (wt/vol %) mannitol.
  • the composition comprises a sugar.
  • the sugar is selected from trehalose, lactose, sucrose, glucose, galactose, maltose, mannose, fructose, dextrose, or a combination thereof.
  • the sugar is selected from trehalose, sucrose, or a combination thereof.
  • the composition comprises from 1% to 40% (wt/vol %) of trehalose, sucrose, or a combination of trehalose and sucrose.
  • the composition comprises from 1% to 20% (wt/vol %) of trehalose, sucrose, or a combination of trehalose and sucrose.
  • the composition comprises 2% (wt/vol %) of trehalose, sucrose, or a combination of trehalose and sucrose.
  • the composition further comprises an osmolyte selected from glycine, carnitine, ethanolamine, their phosphates, mono sugars, or a combination thereof.
  • compositions are isotonic. In other aspects, the compositions are essentially isotonic.
  • the ionic strength of the composition is less than 50 mM. In other aspects, the ionic strength of the composition is less than 10 mM.
  • Antimicrobial agents in bacteriostatic or fungistatic concentrations are typically added to preparations packaged in multiple dose containers which include by way of example and without limitation, phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Other antimicrobial agents are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • Buffers include by way of example and without limitation, acetate, ammonium sulfate, ammonium hydroxide, arginine, aspartic acid, benzene sulfonic acid, benzoate sodium, benzoate acid, carbonate, sodium carbonate, carbon dioxide, citrate, diethanolamine, glucono delta lactone, glycine, glycine HCl, histidine, histidine HCl, hydrochloric acid, hydrobromic acid, lysine maleic acid, meglumine, methanesulfonic acid, monoethanolamine, phosphate, sodium phosphate, citrate, succinate sodium, sulfuric acid, tartarate sodium, trmethamine, sodium citrate, hydroxide, sodium hydroxide, Tris base, Tris base ⁇ 65, Tris acetate, Tris HCl, and Tris HCl-65.
  • the pharmaceutically acceptable carrier comprises a buffer.
  • the buffer is selected from tris, HEPES, histidine, ethylene diamine, or a combination thereof. In other aspects, the buffer is selected from tris, histidine, or a combination thereof.
  • the buffer comprises histidine, which is optionally L-histidine.
  • the composition comprises at least 100 mM histidine. In further aspects, the composition comprises at least 50 mM histidine. In some aspects, the composition comprises at least 20 mM histidine. In additional aspects, the composition comprises 10 to 100 mM histidine. In other aspects, the composition comprises 10 to 20 mM histidine.
  • Antioxidants include by way of example and without limitation, sodium bisulfate, acetone sodium bisulfate, argon, ascorbyl palmitate, ascorbate sodium, ascorbate acid, butylated hydroxy anisole, butylated hydroxy toluene, cysteine, cystenate HCl, dithionite sodium, gentistic acid, gentistic acid ethanoloamine, glutamate monosodium, glutathione, formaldehyde solfoxylate sodium, metabisulfite potassium, metabisulfite sodium, methionine, monothioglycerol, nitrogen, propyl gallate, sulfite sodium, tocopherol alpha, alpha tocopherol hydrogen succinate and thioglycolyate sodium.
  • compositions comprise an antioxidant, a free radical scavenger, a quencher, an antioxidant synergist or a combination thereof.
  • the antioxidant is selected from methionine, butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, or a combination thereof. In other aspects, the antioxidant comprises methionine. In further aspects, the antioxidant is L-methionine. In certain aspects, the compositions comprise at least 20 mM methionine. In other aspects, aspects, the compositions comprise at least 10 mM methionine.
  • Suspending, emulsifying and/or dispersing agents include by way of example and without limitation, sodium carboxymethylcelluose, hydroxypropyl methylcellulose, Polysorbate 80 (TWEEN® 80) and polyvinylpyrrolidone.
  • the compositions comprise a surfactant.
  • the surfactant is selected from polysorbate 20, polysorbate 80, a pluronic, polyoxyethylene sorbitan mono-oleate, polyethylene mono-laureate, N-actylglucoside, or a combination thereof.
  • the surfactant is polysorbate 20.
  • the compositions comprise from 0.0001% to 0.1% (wt/vol %) polysorbate 20.
  • the compositions comprise cyclodextrin.
  • the cyclodextrin comprises (2-hydroxypropyl)- ⁇ -cyclodextrin.
  • a sequestering or chelating agent of metal ions include by way of example and without limitation, calcium disodium EDTA, disodium EDTA, sodium EDTA, calcium versetaminde sodium, calteridol and DPTA.
  • the present compositions comprise a metal chelator.
  • the metal chelator is selected from EDTA, deferoxamine mesylate, EGTA, fumaric acid, and malic acid, salts thereof, or combinations thereof.
  • the metal chelator comprises EDTA or salts thereof.
  • the compositions have an EDTA concentration of about 0.1 mg/ml to about 1.0 mg/ml.
  • isotonic agents buffers, antioxidants, anesthetics, suspending and dispersing agents, emulsifying agents and chelating agents are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • Pharmaceutical carriers also include, by way of example and without limitation, ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid.
  • Other pharmaceutical carriers are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • the chlorotoxin conjugates described herein are often formulated using a variety of parameters including by way of example and without limitation, pH, molarity, % weight/volume, % volume/volume and the like.
  • Other factors considered in the formulation of, stability of, storage of, shipping of chlorotoxin conjugates include by way of example and without limitation, the gas environment, container material, container color, cap material, cap color, presence of additional aspects, such as antioxidants, stabilizers, photoprotective compounds, protectants, sugars, ion chelators, ion donors or the like. Any factor which serves as any one of the above factors known to one of ordinary skill in the art is often used with the chlorotoxin conjugates described herein but not limited as such.
  • compositions are known to those of skill in the art in light of the present disclosure.
  • General techniques for formulation and administration are found in “Remington: The Science and Practice of Pharmacy, Twentieth Edition,” Lippincott Williams & Wilkins, Philadelphia, Pa. Tablets, capsules, pills, powders, granules, dragees, gels, slurries, ointments, solutions suppositories, injections, inhalants and aerosols are examples of such formulations.
  • the chlorotoxin conjugates are often stored at various temperatures, including by way of example and without limitation, freezing, for example at about ⁇ 20° C., about ⁇ 70° C., about ⁇ 100° C., about ⁇ 120° C., about ⁇ 150° C., about ⁇ 200° C. or more than about ⁇ 200° C., cold storage, for example at about 10° C., about 5° C., about 4° C., about 2° C., about 0° C., about ⁇ 2° C. or more than about ⁇ 5° C., or any other suitable temperature such that the composition remains stable.
  • freezing for example at about ⁇ 20° C., about ⁇ 70° C., about ⁇ 100° C., about ⁇ 120° C., about ⁇ 150° C., about ⁇ 200° C. or more than about ⁇ 200° C.
  • cold storage for example at about 10° C., about 5° C., about 4° C., about 2° C., about 0° C.
  • compositions comprising the compounds described herein are stored as lyophilized solids.
  • the present disclosure provides methods for producing the lyophilized composition, the method comprising providing the composition; and lyophilizing the composition, thereby producing the lyophilized composition.
  • lyophilization it is possible to store the compounds in a manner that maintains physiological or otherwise optimal pH, isotonicity and stability.
  • materials include pH buffers, preservatives, tonicity adjusting agents, anti-oxidants, other polymers (e.g., viscosity adjusting agents or extenders) and excipients to stabilize the labile protein against the stresses of drying and storage of the dried product.
  • additives include phosphate, citrate, or borate buffers; thimerosal; sorbic acid; methyl or propyl paraben, and chlorobutanol preservatives; sodium chloride: polyvinyl alcohol, polyvinyl pyrrolidone; mannitol, dextrose, dextran, lactose, sucrose, ethylene diamine tetra-acetic acid, and the like.
  • Suitable formulations known in the art, (Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton, Pa.; Arakawa et al. (1990), supra; Carpenter et al. (1991), supra; and Pikal (1990), supra).
  • the pharmaceutically acceptable carrier comprises a reconstitution stabilizer.
  • the reconstitution stabilizer comprises a water-soluble polymer.
  • the water-soluble polymer is selected from a polaxamer, a polyol, a polyethylene glycol, a polyvinylalcohol, a hydroxyethyl starch, dextran, polyvinylpyrrolidene poly(acrylic acid), or a combination thereof.
  • substitution stabilizer means any excipient which is capable of preventing aggregation of a reconstituted protein in an aqueous medium.
  • Excipients possessing the necessary characteristics for the present invention are well-known in the art and generally function by the mechanisms of charge replusion, steric hindrance, hydrophobic binding or specific high-affinity binding to the dried protein.
  • Exemplary excipients include various osmolytes, various salts, water soluble synthetic and natural polymers, surfactants, sulfated polysaccharides, carrier proteins, buffers and the like (Manning et al. (1989), Pharmaceutical Research, 6:903-918; and Paborji, et al. (1994), Pharmaceutical Research, 11:764-771).
  • the present compounds and an effective amount of the reconstitution stabilizer are admixed under conditions effective to reduce aggregation of present compounds upon reconstitution with the reconstitution medium (e.g., a solvent and optionally other components such as antibacterials).
  • the reconstitution stabilizer may be admixed with the compouns at a suitable time before, during or after reconstitution; preferably the reconstitution stabilizer will be pre-dissolved in the reconstitution medium.
  • the compound is reconstituted at a temperature which is above the freezing point of the reconstitution medium, but which will not degrade the compound and which will not be deleterious to the reconstitution stabilizer; preferably the temperature will be between about 2° C. to 50° C.
  • the time taken to mix the reconstitution stabilizer and the dried compound should be for a sufficient period to prepare a suitable admixture; preferably mixing will be for between about 1 to 30 minutes. Generally, the reconstituted formulation is used soon after reconstitution.
  • the present compositions are reconstituted from a lyophilized form.
  • the present disclosure provides methods for producing the reconstituted composition, the method comprising providing a lyophilized composition; and reconstituting the composition with a solution to produce a reconstituted composition.
  • the reconstituting solution comprises water.
  • the reconstituting solution is selected from sterile water, physiological saline solution, glucose solution or other aqueous solvents (e.g., alcohols such as ethyl, n-propyl or isopropyl, butyl alcohol), or a combination thereof, which are capable of dissolving the dried composition and compatible with the selected administration route and which does not negatively interfere with the compound and the reconstitution stabilizers employed.
  • aqueous solvents e.g., alcohols such as ethyl, n-propyl or isopropyl, butyl alcohol
  • the chlorotoxin conjugates are placed into containers following formulation.
  • the containers include by way of example and without limitation, glass, for example amber glass or colorless glass.
  • the containers often include by way of example and without limitation, a plastic, a rubber, a metal, a biodegradable material or the like known to one of skill in the art and are any color, colorless, opaque or clear.
  • the chlorotoxin conjugates are placed into containers which are sealed following formulation. Often the seal is a cap, for example, the cap is a plastic, a rubber, a metal, a biodegradable material, a combination of or the like known to one of skill in the art and are any color, colorless, opaque or clear, sometimes a film.
  • a gas is included in the container, often to enhance stability of the chlorotoxin conjugate or prevent oxygen from contacting the chlorotoxin conjugate.
  • gas includes by way of example and without limitation, an inert gas, such as nitrogen or argon, occasionally a noble gas and is used at any suitable concentration.
  • the compounds of the present disclosure are stored in a vessel comprising glass, particularly a Type I glass, which has been subjected to a washing or extraction treatment which reduces the level of extractable trivalent and divalent metal ions present in/on the surface of the glass.
  • a washing or extraction treatment which reduces the level of extractable trivalent and divalent metal ions present in/on the surface of the glass.
  • Such treatments include steeping in (extraction with) hot (preferably at least 90° C.) water or another aqueous medium, e.g. ammonium sulfate solution, or treatment with sulfur dioxide.
  • the compounds of the present disclosure are stored in a vessel comprising USP Type 1 borosilicate glass vial with a 13 mm chlorobutyl based stopper with flourotech coating on plug and B2 coating on the top and an aluminum over seal with flip top cap.
  • the compounds of the present disclosure are stored in a foil-lined chamber.
  • the chamber comprises a dry inert atmosphere, preferably nitrogen, and a desiccant or oxygen absorber.
  • the present disclosure provides a kit comprising vessel configured to contain a fluid; any of the compounds and compositions described herein; and an elastomeric closure affixed to the vessel.
  • the kit further comprises a light shield.
  • the light shield is a physical barrier configured to block at least a portion of the light incident on the vessel from the composition.
  • the physical barrier comprises an opaque or semi-opaque material.
  • the vessel is a glass vial.
  • the glass vial comprises clear or amber glass.
  • the glass vial is an untreated glass container.
  • the glass vial comprises USP Type I, Type II, Type III, or Type IV glass.
  • the inner portion of the vessel further comprises a silica (SiO 2 ) coating or silicone coating.
  • the untreated glass container is selected from an ampoule, vial, ready-to-use syringe, or carpoule.
  • the elastomeric closure is a halobutyl rubber closure.
  • the halobutyl rubber closure is selected from a chlorobutyl rubber closure or a bromobutyl rubber closure.
  • elastomeric closure is coated with Fluorotec, B2, or a combination thereof.
  • the kit further comprises an opaque secondary package surrounding the vessel.
  • the opaque secondary package comprises an opaque box, an opaque aluminum foil pouch, or a combination thereof.
  • the opaque secondary package is configured to block at least 90% of the light incident on the package exterior from the composition.
  • the opaque secondary package is configured to block at least 95% of the light incident on the package exterior from the composition.
  • the opaque secondary package is configured to block at least 99% of the light incident on the package exterior from the composition.
  • the opaque secondary package is configured to block at least 99.9% of the light incident on the package exterior from the composition.
  • the vessel comprises a reduced-oxygen environment in contact with the composition.
  • the vessel comprises an inert gas in contact with the composition.
  • the composition is sparged with an inert gas, thereby producing the reduced-oxygen environment in the vessel.
  • the inert gas comprises nitrogen or argon.
  • the product or dosage form characteristics which result from the processing methods and/or parameters for generating formulations such as powders, lyophilized compositions, and the like include, but are not limited to, density, water content, friability, disintegration, dissolution profile(s), shape, size, weight, uniformity and composition of the particles. These product characteristics are often modulated in a number of ways and affect the final in vitro and/or in vivo performance of the formulations. Product or dosage form characteristics are often a consequence of excipient selection, excipient composition, manufacturing methods applied or a combination of any of these. The combination of excipients as well as product characteristics (including processing methods or processing parameters) of the final dosage form will ultimately determine the pharmacokinetic profile of the active ingredient in vivo.
  • the administered chlorotoxin conjugate formulations described herein are often processed or manufactured under specific conditions such as, for example, mixing methods (including sieve size, rpm, and milling), drying time, conditions, environmental parameters (e.g., temperature and humidity) and combinations thereof) which themselves modulate the pharmacokinetic profile of chlorotoxin compositions in vivo (i.e., increase the average C max or AUC).
  • mixing methods including sieve size, rpm, and milling
  • drying time conditions
  • environmental parameters e.g., temperature and humidity
  • environmental parameters e.g., temperature and humidity
  • Dissolution and drug release from formulations depends on many factors including the solubility and concentration of the active ingredient, the nature and composition of the excipients, content uniformity, water content, product shape and size, porosity, disintegration time and other factors.
  • the release of a drug or active ingredient from a final dosage form in vitro is typically characterized by its dissolution profile under standardized conditions (using United States Pharmacopeia (USP) or similar accepted methods for reference) and at the appropriate pH, often a neutral pH.
  • USP United States Pharmacopeia
  • Standard conditions make use of buffers at an appropriate pH in order to best mimic the pH of a subject's blood.
  • a therapeutically effective dosage is formulated to contain a dose of at least about 0.1 mg up to about 1.5 mg or more, such as more than 1.5 mg of chlorotoxin conjugate.
  • the effective dosage is formulated to contain a dose of at least about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.5 mg, about 0.07 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.35 mg, about 0.375 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.8 mg, about 1.9 mg, about 2 mg, about 2.4 mg, about 3 mg, about 5 mg, about 6 mg, about 7 mg, about 12 mg, about 18 mg or about 60 mg more of chlorotoxin conjugate.
  • the dose is 0.1 mg for a mouse, 1 mg for a dog, 0.3 mg for a rat, 0.6 mg for a monkey and 3 mg for a human.
  • the amount of chlorotoxin conjugate administered to a subject is often the total about amount listed herein. In some aspects, the amount of chlorotoxin conjugate administered to a subject is often the about per milligram, gram or kilogram of subject weight for each amount listed herein. In other aspects, the amount of chlorotoxin conjugate administered to a subject is often the about per milliliter or liter of fluid volume for each amount listed herein. In yet other aspects, the amount of chlorotoxin conjugate administered to a subject is often the about per square millimeter, square centimeter or square meter of subject surface body area or subject body area for each amount listed herein.
  • a “dosage regimen” refers to the protocol used to administer an intravenous pharmaceutical formulation comprising chlorotoxin conjugate to a subject.
  • the dosage regimen comprises a dose amount and dosing interval.
  • the dosage regimen further comprises a dosing duration.
  • dosing duration refers to the period of time over which a dose is administered.
  • the dose of chlorotoxin conjugate is administered to a subject using either a fixed or a scaling dosing scheme.
  • a fixed dosing scheme includes administration of a bolus or continuous dose of chlorotoxin conjugate to a subject via an intravenous administration route wherein the fixed dose is, for example and without limitation, about 3 mg to about 6 mg and does not account or adjust for a subject's age, weight, height, body mass index, metabolism, or the like.
  • a scaling dosing scheme includes administration of a bolus or continuous dose of chlorotoxin conjugate to a subject via an intravenous administration route wherein the scaled dose is, for example and without limitation, about 3 mg to about 6 mg and accounts or adjusts for a subject's age, weight, height, body mass index, metabolism, or the like.
  • the fixed dose and/or the scaled dose are determined for one subject based upon the dose administered to a different subject wherein the subjects are or are not the same species, for example a mouse and a human or a rat and a human, or a dog and a human or a monkey and a human or a non-human primate and a human.
  • the same dose or about the same dose is administered to all subjects, for example a mouse and a human or a rat and a human, or a dog and a human or a monkey and a human or a non-human primate and a human.
  • the scaled dose to be administered to a subject is determined from the dose administered to a different subject wherein the subjects are or are not the same species, for example a mouse and a human or a rat and a human, or a dog and a human or a monkey and a human or a non-human primate and a human.
  • the scaled dose is therefore increased from the dose administered to the mouse, rat, dog, monkey or non-human primate to the dose administered to the human based upon the difference between the mouse, rat, dog, monkey or non-human primate and the human on factors such as subject age, weight, height, metabolism, size or the like.
  • the dose is scaled from a rat to a human.
  • compositions of chlorotoxin conjugates are administered to a subject before surgery, during surgery and/or the excised tissue from the subject is contacted with compositions of the chlorotoxin conjugates.
  • compositions of chlorotoxin conjugates are intravenously administered to a subject about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 9 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours or about 72 hours before surgery.
  • compositions of chlorotoxin conjugates are intravenously administered to a subject between 0 and 1 hours, between 1 and 2 hours, between 2 and 3 hours, between 3 and 4 hours, between 4 and 5 hours, between 5 and 6 hours, between 6 and 9 hours, between 9 and 12 hours, between 12 and 24 hours, between 24 and 36 hours, between 36 and 48 hours or between 48 and 72 hours (inclusive) before surgery.
  • Tissue or fluid samples are often isolated from a subject prior to administration of a chlorotoxin conjugate, sometimes as a baseline reference.
  • Samples are also isolated from a subject after administration of the compounds of the present disclosure, often less than about 1 minute after, about 2 minutes after, about 3 minutes after, about 4 minutes after, about 5 minutes after, about 6 minutes after, about 7 minutes after, about 8 minutes after, about 9 minutes after, about 10 minutes after, about 11 minutes after, about 12 minutes after, about 13 minutes after, about 14 minutes after, about 15 minutes after, about 20 minutes after, about 30 minutes after, about 40 minutes after, about 50 minutes after, about 60 minutes after, about 1 hour after, about 2 hours after, about 3 hours after, about 4 hours after, about 5 hours after, about 6 hours after, about 12 hours after, about 18 hours after, about 24 hours after, about 36 hours after, about 48 hours after, about 72 hours after, about 96 hours after, about 5 days after, about 7 days after, about 10 days after, about 14 days after, about 21 days after, about 4 weeks after, about 6 weeks after, about 8 weeks after, about 12 weeks after, about 16 weeks after, about 20 weeks after or more than 20 weeks after.
  • the methods and compositions described herein relate to pharmacokinetics of intravenous administration of chlorotoxin conjugates to a subject.
  • Pharmacokinetics are often described using models, for example, compartmental or noncompartmental models.
  • Compartmental models include but are not limited to monocompartmental model, the two compartmental model, the multicompartmental model or the like. Models are often divided into different compartments and described by the corresponding scheme. For example, one scheme is the absorption, distribution, metabolism and excretion (ADME) scheme. For another example, another scheme is the liberation, absorption, distribution, metabolism and excretion (LADME) scheme. In some aspects, metabolism and excretion are grouped into one compartment referred to as the elimination compartment.
  • ADME absorption, distribution, metabolism and excretion
  • LADME liberation, absorption, distribution, metabolism and excretion
  • metabolism and excretion are grouped into one compartment referred to as the elimination compartment.
  • liberation includes liberation of the active portion of the composition from the delivery system
  • absorption includes absorption of the active portion of the composition by the subject
  • distribution includes distribution of the composition through the blood plasma and to different tissues
  • metabolism which includes metabolism or inactivation of the composition
  • excretion which includes excretion or elimination of the composition or the products of metabolism of the composition.
  • compositions administered intravenously to a subject are subject to multiphasic absorption including but not limited to aspects of tissue distribution and metabolism/excretion.
  • the decrease in plasma concentration of the composition is often biphasic, including, for example an alpha phase and a beta phase, occasionally a gamma, delta or other phase is observed.
  • the bioavailability of the compositions described herein is absolute bioavailability, often 1 or 100% given intravenous administration.
  • Pharmacokinetics includes determining at least one parameter associated with intravenous administration of chlorotoxin conjugates to a subject.
  • parameters include at least the dose (D), dosing interval ( ⁇ ), area under curve (AUC), maximum concentration (C max ), minimum concentration reached before a subsequent dose is administered (C min ), minimum time (Tin), maximum time to reach C max (T max ), volume of distribution (V d ), back-extrapolated concentration at time 0 (C 0 ), steady state concentration (C ss ), elimination rate constant (k e ), infusion rate (k in ), clearance (CL), bioavailability (f), fluctuation (% PTF) and elimination half-life (T 1/2 ).
  • the C max , C 0 , and AUC increase in a dose-dependent manner.
  • the compounds described herein have values for at least one of the pharmacokinetic parameters listed herein and known to those of ordinary skill in the art. Often, the values for the pharmacokinetic parameters are recorded, observed, measured, processed, analyzed or the like as data.
  • the pharmacokinetics parameters are any parameters suitable for describing the plasma or serum profiles of chlorotoxin conjugates described herein.
  • the pharmacokinetics profile are often obtained at a time after dosing of, for example, about zero minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about
  • the pharmacokinetics parameters are any parameters suitable for describing the plasma or serum profiles of chlorotoxin conjugates described herein.
  • the dose (D) includes by way of example but is not limited to, about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.5 mg, about 0.07 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.35 mg, about 0.375 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.8 mg, about 1.9 mg, about 2 mg, about 2.4 mg, about 3 mg, about 5 mg, about 6 mg, about 7 mg, about 12 mg, about 18 mg or about 60 mg more of chlorotoxin conjugate.
  • the dosing interval ( ⁇ ) includes by way of example but is not limited to, about 12 hours, about 24 hours, about 36 hours, about 48 hours or about 72 hours before surgery.
  • the pharmacokinetics parameters are any parameters suitable for describing the plasma or serum profiles of chlorotoxin conjugates described herein.
  • the area under curve (AUC) includes by way of example but is not limited to, not less than about 50 hr*ng/mL, not less than about 75 hr*ng/mL, not less than about 100 hr*ng/mL, not less than about 125 hr*ng/mL, not less than about 150 hr*ng/mL, not less than about 175 hr*ng/mL, not less than about 200 hr*ng/mL, not less than about 250 hr*ng/mL, not less than about 300 hr*ng/mL, not less than about 350 hr*ng/mL, not less than about 400 hr*ng/mL, not less than about 500 hr*ng/mL, not less than about 600 hr*ng/mL, not less than about 700 hr*ng/mL, not less than
  • the AUC of a chlorotoxin described herein by way of example can be, but is not limited to, about 1,000 hr*ng/mL to about 1,250 hr*ng/mL; about 1,250 hr*ng/mL to about 1,500 hr*ng/mL; about 1,500 hr*ng/mL to about 1,750 hr*ng/mL; about 1,750 hr*ng/mL to about 2,000 hr*ng/mL; about 2,000 hr*ng/mL to about 2,500 hr*ng/mL; about 2,500 hr*ng/mL to about 3,000 hr*ng/mL; about 3,000 hr*ng/mL to about 3,500 hr*ng/mL; about 3,500 hr*ng/mL to about 4,000 hr*ng/mL; about 4,000 hr*ng/mL to about 4,500 hr*ng/mL; about 4,500 hr*ng/mL to
  • the pharmacokinetic parameters is any parameters suitable for describing a chlorotoxin conjugate described herein.
  • the C max includes by way of example but is not limited to not less than about 1 ng/mL; not less than about 5 ng/mL; not less than about 10 ng/mL; not less than about 15 ng/mL; not less than about 20 ng/mL; not less than about 25 ng/mL; not less than about 50 ng/mL; not less than about 75 ng/mL; not less than about 100 ng/mL; not less than about 200 ng/mL; not less than about 300 ng/mL; not less than about 400 ng/mL; not less than about 500 ng/mL; not less than about 600 ng/mL; not less than about 700 ng/mL; not less than about 800 ng/mL; not less than about 900 ng/mL; not less than about 1000 ng/mL; not less than about 1250 ng/mL; not less
  • the C max is, for example, about 1 ng/mL to about 100,000 ng/mL; about 1 ng/mL to about 95.00 ng/mL; about 1 ng/mL to about 90,000 ng/mL; about 1 ng/mL to about 8500 ng/mL; about 1 ng/mL to about 80000 ng/mL; about 1 ng/mL to about 7500 ng/mL; about 1 ng/mL to about 70,000 ng/mL; about 1 ng/mL to about 65 00 ng/mL; about 1 ng/mL to about 60,000 ng/mL; about 1 ng/mL to about 55000 ng/mL; about 1 ng/mL to about 50000 ng/mL; about 1 ng/mL to about 40000 ng/mL; about 1 ng/mL to about 30000 ng/mL; about 1 ng/mL to about 20000 ng/mL; about 1 ng/mL
  • the plasma concentration of a chlorotoxin conjugate described herein includes by way of example but is not limited to, not less than about 1 ng/mL, not less than about 2 ng/mL, not less than about 3 ng/mL, not less than about 4 ng/mL, not less than about 5 ng/mL, not less than about 6 ng/mL, not less than about 7 ng/mL, not less than about 8 ng/mL, not less than about 9 ng/mL, not less than about 10 ng/mL, not less than about 11 ng/mL, not less than about 12 ng/mL, not less than about 13 ng/mL, not less than about 14 ng/mL, not less than about 15 ng/mL, not less than about 16 ng/mL, not less than about 17 ng/mL, not less than about 18 ng/mL, not less than about 19 ng/mL, not less than about 20 ng/mL, not less than about 21 ng
  • the plasma concentration includes by way of example but is not limited to, about 1 ng/mL to about 2 ng/mL; about 1 ng/mL to about 5 ng/mL; about 5 ng/mL to about 10 ng/mL; about 10 ng/mL to about 25 ng/mL; about 25 ng/mL to about 50 ng/mL; about 50 ng/mL to about 75 ng/mL; about 75 ng/mL to about 100 ng/mL; about 100 ng/mL to about 150 ng/mL; about 100 ng/mL to about 200 ng/mL about 150 ng/mL to about 200 ng/mL; about 200 ng/mL to about 250 ng/mL; about 250 ng/mL to about 300 ng/mL; about 300 ng/mL to about 350 ng/mL; about 350 ng/mL to about 400 ng/mL; about 400 ng/mL to about 450 ng/mL;
  • the T max of a chlorotoxin conjugate described herein includes by way of example but is not limited to, not greater than about 0.5 minutes, not greater than about 1 minutes, not greater than about 1.5 minutes, not greater than about 2 minutes, not greater than about 2.5 minutes, not greater than about 3 minutes, not greater than about 3.5 minutes, not greater than about 4 minutes, not greater than about 4.5 minutes, not greater than about 5 minutes, or any other T max appropriate for describing a pharmacokinetic profile of a chlorotoxin conjugate described herein.
  • the T max further includes by way of example but is not limited to about 0.1 minutes to about 24 minutes; about 0.1 minutes to about 0.5 minutes; about 0.5 minutes to about 1 minute; about 1 minute to about 1.5 minutes; about 1.5 minutes to about 2 minute; about 2 minutes to about 2.5 minutes; about 2.5 minutes to about 3 minutes; about 3 minutes to about 3.5 minutes; about 3.5 minutes to about 4 minutes; about 4 minutes to about 4.5 minutes; about 4.5 minutes to about 5 minutes; about 5 minutes to about 5.5 minutes; about 5.5 minutes to about 6 minutes; about 6 minutes to about 6.5 minutes; about 6.5 minutes to about 7 minutes; about 7 minutes to about 7.5 minutes; about 7.5 minutes to about 8 minutes; about 8 minutes to about 8.5 minutes; about 8.5 minutes to about 9 minutes; about 9 minutes to about 9.5 minutes; about 9.5 minutes to about 10 minutes; about 10 minutes to about 10.5 minutes; about 10.5 minutes to about 11 minutes; about 11 minutes to about 11.5 minutes; about 11.5 minutes to about 12 minutes; about 12 minutes to about 12.5 minutes; about 12.5 minutes to about 13 minutes;
  • the T max of a chlorotoxin conjugate described herein includes by way of example but is not limited to, not greater than about 0.5 hours, not greater than about 1 hours, not greater than about 1.5 hours, not greater than about 2 hours, not greater than about 2.5 hours, not greater than about 3 hours, not greater than about 3.5 hours, not greater than about 4 hours, not greater than about 4.5 hours, not greater than about 5 hours, or any other T max appropriate for describing a pharmacokinetic profile of a chlorotoxin conjugate described herein.
  • the T max further includes by way of example but is not limited to about 0.1 hours to about 24 hours; about 0.1 hours to about 0.5 hours; about 0.5 hours to about 1 hour; about 1 hour to about 1.5 hours; about 1.5 hours to about 2 hour; about 2 hours to about 2.5 hours; about 2.5 hours to about 3 hours; about 3 hours to about 3.5 hours; about 3.5 hours to about 4 hours; about 4 hours to about 4.5 hours; about 4.5 hours to about 5 hours; about 5 hours to about 5.5 hours; about 5.5 hours to about 6 hours; about 6 hours to about 6.5 hours; about 6.5 hours to about 7 hours; about 7 hours to about 7.5 hours; about 7.5 hours to about 8 hours; about 8 hours to about 8.5 hours; about 8.5 hours to about 9 hours; about 9 hours to about 9.5 hours; about 9.5 hours to about 10 hours; about 10 hours to about 10.5 hours; about 10.5 hours to about 11 hours; about 11 hours to about 11.5 hours; about 11.5 hours to about 12 hours; about 12 hours to about 12.5 hours; about 12.5 hours to about 13 hours;
  • the chlorotoxin conjugates distribute into the subject tissues. For example, distribution into the tissues is often rapid compared to the elimination phase. In some aspects, the chlorotoxin conjugates are eliminated from the subject tissues. For example, elimination from the subject tissues is often slow compared to the distribution phase. Often the kidney is important in the clearance and elimination of the chlorotoxin conjugates, often contributing to the elimination phase.
  • the pharmacokinetics parameters are any parameters suitable for describing the plasma profiles of chlorotoxin conjugates described herein and are often associated with a curve.
  • dose is either scaled or fixed, said scaled dose useful for scaling the dose from one subject to another wherein the subjects are the same species, different species, same sex or different sex.
  • the phases of the curve are often representative of data obtained from at least one subject, sometimes more than one subject, and the phases of the curve and/or data of the curve is often scaled in a manner similar to the manner in which doses are scaled.
  • the curve is plotted on a graph, often a graph with an x-axis and a y-axis referred to for example as an x-y plot, a scatter plot or the like.
  • Each axis of the graph has units, the y-axis often having units of time, for example in hours, and x-axis often having units of concentration, for example as ng/mL, of a chlorotoxin conjugate described herein present in a subject sample as described herein and are representative of a single measurement, a mean, an average, or any other suitable mathematical calculation performed on a set of data.
  • a statistic is also calculated, for example, a standard error, standard error of the mean, standard deviation, standard deviation of the mean, or any other suitable statistic useful for the described disclosure.
  • the curve has phases, for example, distribution phase, metabolism phase and elimination phase.
  • the distribution phase begins at time of about 0 hours and extends until a time of about 0.01 hours, about 0.02 hours, about 0.03 hours, about 0.04 hours, about 0.05 hours, about 0.06 hours, about 0.07 hours, about 0.08 hours, about 0.09 hours, about 0.11 hours, about 0.12 hours, about 0.13 hours, about 0.14 hours, about 0.15 hours, about 0.16 hours, about 0.17 hours, about 0.18 hours, about 0.19 hours, about 0.20 hours, 0.21 hours, about 0.22 hours, about 0.23 hours, about 0.24 hours, about 0.25 hours, about 0.26 hours, about 0.27 hours, about 0.28 hours, about 0.29 hours, about 0.30 hours, about 0.31 hours, about 0.32 hours, about 0.33 hours, about 0.34 hours, about 0.35 hours, about 0.36 hours, about 0.37 hours, about 0.38 hours, about 0.39 hours, about 0.40 hours, about 0.41 hours, about 0.42 hours, about 0.43 hours, about 0.
  • the metabolism phase begins at time of about 0.5 hours and extends until a time of about 0.50 hours, about 0.51 hours, about 0.52 hours, about 0.53 hours, about 0.54 hours, about 0.55 hours, about 0.56 hours, about 0.57 hours, about 0.58 hours, about 0.59 hours, about 0.60 hours, about 0.61 hours, about 0.62 hours, about 0.63 hours, about 0.64 hours, about 0.65 hours, about 0.66 hours, about 0.67 hours, about 0.68 hours, about 0.69 hours, about 0.70 hours, about 0.71 hours, about 0.72 hours, about 0.73 hours, about 0.74 hours, about 0.75 hours, about 0.76 hours, about 0.77 hours, about 0.78 hours, about 0.79 hours, about 0.80 hours, about 0.81 hours, about 0.82 hours, about 0.83 hours, about 0.84 hours, about 0.85 hours, about 0.86 hours, about 0.87 hours, about 0.88 hours, about 0.89 hours, about 0.90 hours, about 0.91 hours, about 0.92 hours, about 0.93 hours, about 0.94 hours, about 0.95 hours, about 0.50 hours,
  • the elimination phase begins at time of about 2 hours and extends until a time of about 2.00 hours, about 2.20 hours, about 2.40 hours, about 2.60 hours, about 2.80 hours, about 3.00 hours, about 4.20 hours, about 4.40 hours, about 4.60 hours, about 4.80 hours, about 5.00 hours, about 5.20 hours, about 5.40 hours, about 5.60 hours, about 5.80 hours, about 6.00 hours, about 6.20 hours, about 6.40 hours, about 6.60 hours, about 6.80 hours, about 7.00 hours, about 7.20 hours, about 7.40 hours, about 7.60 hours, about 7.80 hours, about 8.00 hours, about 8.20 hours, about 8.40 hours, about 8.60 hours, about 8.80 hours, about 9.00 hours, about 9.20 hours, about 9.40 hours, about 9.60 hours, about 9.80 hours, about 10.00 hours, about 10.20 hours, about 10.40 hours, about 10.60 hours, about 10.80 hours, about 12.00 hours, about 12.20 hours, about 12.40 hours, about 12.60 hours, about 12.80 hours, about 14.00 hours, about 14.20
  • a single fixed bolus dose intravenous chlorotoxin conjugate often results in mean serum concentrations measurable up to about 12 hours post-dose, about 24 hours post-dose, up to about 36 hours post-dose, up to about 48 hours post-dose or more than about 48 hours post-dose.
  • C max , and C 0 parameters increase in about a dose-proportional manner.
  • the AUC 0-t parameter, for subjects such as rats is about dose-proportional at less than about a 1 mg dose levels, and increases in a greater than dose-proportional manner at greater than about 1 mg dose levels.
  • PK parameters are predictive in rats of a human subject.
  • a single fixed bolus dose intravenous chlorotoxin conjugate often results in mean serum concentrations measurable up to about 12 hours post-dose, about 24 hours post-dose, up to about 36 hours post-dose, up to about 48 hours post-dose or more than about 48 hours post-dose.
  • C max and C 0 parameters increase in about a dose-proportional manner.
  • the AUC 0-t parameter for subjects such as monkeys, is greater than dose-proportional manner at greater than about 1 mg dose levels for example such that chlorotoxin conjugates exhibit reduced clearance at higher doses in monkeys.
  • the C 0 and AUC are about 5 to about 30% higher in females relative to males.
  • two pharmacokinetic profiles are “about equivalent” if they are defined by at least one parameter that is about equivalent between the two profiles.
  • Non-limiting examples of such parameters include the area under plasma concentration over time curve (AUC) and the maximal plasma concentration reached following administration of a dose (C max ).
  • two pharmacokinetic parameters are about equivalent if the lower value is greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% of the higher value.
  • the pharmacokinetic profiles of two dosage regimens are compared by determining the average pharmacokinetic profile in a population of subjects receiving the first dosage regimen, determining the average pharmacokinetic profile in a population of subjects receiving the second dosage regimen, and then comparing those two population dosage regimens.
  • a population of subjects is one subject.
  • a population of subjects is more than one subject, for example, two subjects, three subjects, four subjects, five subjects, six subjects, seven subjects, eight subjects, nine subjects, ten subjects, 11 subjects, 12 subjects, 13 subjects, 14 subjects, 15 subjects, 20 subjects, 25 subjects, 30 subjects, 35 subjects, 40 subjects, 45 subjects, 50 subjects, or more than 50 subjects.
  • the present disclosure provides a method of administering a composition to a human subject, the method comprising: intravenously administering to the human subject a dose of from 1 mg to 30 mg of a compound comprising a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof; and producing in the human subject an average maximum compound blood plasma concentration (average C max ) of at least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of the compound administered.
  • average C max average maximum compound blood plasma concentration
  • the present disclosure provides a method of detecting a cancer cell in a human subject, the method comprising: intravenously administering to the human subject a dose of from 1 mg to 30 mg of a compound comprising a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof conjugated to a detectable label; producing in the human subject an average maximum compound blood plasma concentration (average C max ) of at least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of the compound administered; and detecting the presence or absence of the detectable label in the human subject, wherein the presence of the detectable label indicates the presence of the cancer cell.
  • average C max average maximum compound blood plasma concentration
  • the present disclosure provides a method of diagnosing cancer in a human subject, the method comprising: intravenously administering to the human subject a dose of from 1 mg to 30 mg of a compound comprising a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof conjugated to a detectable label; producing in the human subject an average maximum compound blood plasma concentration (average C max ) of at least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of the compound administered; and detecting the presence or absence of the detectable label in the human subject, wherein the presence of the detectable label indicates a diagnosis of cancer.
  • the present disclosure provides a method of treating cancer in a human subject, the method comprising: intravenously administering to the human subject a dose of from 1 mg to 30 mg of a compound comprising a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof conjugated to a therapeutic agent; producing in the human subject an average maximum compound blood plasma concentration (average C max ) of at least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of the compound administered; and reducing or improving a symptom or condition associated with cancer in the human subject.
  • the human subject is in need thereof.
  • the methods comprise administering a therapeutically effective dose of the compound to the human subject.
  • the present disclosure provides a method of administering a composition to a human subject, the method comprising: administering to the human subject a dose of from 1 mg to 30 mg of a compound comprising a polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof; and producing in the human subject pharmacokinetic profile of FIG. 27 .
  • the present disclosure provides a method of administering a composition to a human subject, the method comprising: intravenously administering to the human subject a dose of from 1 mg to 30 mg of any suitable compound of the present disclosure; and producing in the human subject an average maximum compound blood plasma concentration (average C max ) of at least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of the compound administered.
  • average C max average maximum compound blood plasma concentration
  • the average time (average T max ) at which the average C max is reached is at 5 ⁇ 4 minutes following administration of the compound. In some aspects, the average time (average T 75 ) at which the average compound blood plasma concentration reaches 75% of the average C max (average C 75 ) is reached is at 8 ⁇ 5 minutes following administration of the compound. In some aspects, the average time (average T 50 ) at which the average compound blood plasma concentration reaches 50% of the average C max (average C 50 ) is reached is at 20 ⁇ 8 minutes following administration of the compound. In some aspects, the average time (average T 25 ) at which the average compound blood plasma concentration reaches 25% of the average C max (average C 25 ) is reached is at 30 ⁇ 12 minutes following administration of the compound.
  • the methods further comprise producing in the human subject an average chlorotoxin polypeptide plasma area under the curve (average AUC) of from 50 hr*ng/mL to 120 hr*ng/mL per each 1 mg dosage of chlorotoxin polypeptide administered.
  • average AUC average chlorotoxin polypeptide plasma area under the curve
  • the methods further comprise producing in the human subject an average chlorotoxin polypeptide plasma area under the curve (average AUC) of from 60 hr*ng/mL to 110 hr*ng/mL per each 1 mg dosage of chlorotoxin polypeptide administered.
  • average AUC average chlorotoxin polypeptide plasma area under the curve
  • 75% of the average AUC occurs within 40 ⁇ 15 minutes after administering the compound. In some aspects, 50% of the average AUC occurs within 21 ⁇ 8 minutes after administering the compound. In some aspects, 25% of the average AUC occurs within 9 ⁇ 5 minutes after administering the compound.
  • the compound comprises any suitable compound of the present disclosure.
  • the present disclosure provides a method for detecting a cancer cell in a subject, the method comprising: administering any suitable compound of the present disclosure; and detecting the presence or absence of the compound in the subject, wherein the presence of the compound indicates the presence of a cancer cell.
  • the method further comprises administering the compound as a part of a composition.
  • the cancer is selected from glioma, astrocytoma, medulloblastoma, choroids plexus carcinoma, ependymoma, brain tumor, neuroblastoma, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, head and neck cancer, lung cancer, breast cancer, intestinal cancer, pancreatic cancer, liver cancer, kidney cancer, sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, carcinoma, melanoma, ovarian cancer, cervical cancer, lymphoma, thyroid cancer, anal cancer, colo-rectal cancer, endometrial cancer, germ cell tumor, laryngeal cancer, multiple myeloma, prostate cancer, retinoblastoma, gastric cancer, testicular cancer, or Wilm's tumor.
  • the cancer is selected from glioma, medulloblastoma, sarcoma, breast cancer, lung cancer, prostate cancer, or intestinal cancer.
  • the cancer cell expresses a site to which native chlorotoxin binds.
  • the method comprises detecting the compound by fluorescence imaging.
  • the method further comprises differentiating a focus of a cancer that expresses a site to which native chlorotoxin binds from non-neoplastic tissue.
  • the method further comprises surgically removing from the subject a cancer cell that is detected.
  • the method further comprises determining the location of a cancer cell in the subject before surgically removing the cancer cell from the subject, during surgical removal of the cancer cell from the subject, after removing the cancer cell from the subject, or a combination thereof.
  • the compound binds to the cancer cell.
  • the subject is a human subject.
  • the detection is performed in vivo or ex vivo.
  • the present disclosure provides a method of administering any suitable compound of the present disclosure to a subject, the method comprising administering a therapeutically effective amount of the compound to the subject.
  • the subject is in need thereof.
  • a therapeutically effective amount is a dosage sufficient for the detection of a cancer cell in the subject.
  • the dosage is from 0.1 mg to 100 mg.
  • dosage is from 1 mg to 30 mg.
  • the dosage is from 3 mg to 30 mg.
  • the present disclosure provides a method of treating a subject in need thereof, the method comprising administering to the subject any suitable compound of the present disclosure further comprising a therapeutic agent in an amount sufficient to treat cancer in the subject.
  • the therapeutic agent is a cytotoxic agent.
  • the cancer is selected from glioma, astrocytoma, medulloblastoma, choroids plexus carcinoma, ependymoma, brain tumor, neuroblastoma, head and neck cancer, lung cancer, breast cancer, intestinal cancer, pancreatic cancer, liver cancer, kidney cancer, sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, carcinoma, melanoma, ovarian cancer, cervical cancer, lymphoma, thyroid cancer, anal cancer, colo-rectal cancer, endometrial cancer, germ cell tumor, laryngeal cancer, multiple myeloma, prostate cancer, retinoblastoma, gastric cancer, testicular cancer, or Wilm's tumor.
  • the cancer cell is selected from glioma, medulloblastoma, sarcoma, prostate cancer, or intestinal cancer.
  • the cancer cell expresses a site to which native chlorotoxin binds. In further aspects, the binding is selective.
  • the compound is administered parenterally. In other aspects, the compound is administered intravenously. In still other aspects, the compound is administered subcutaneously.
  • samples are analyzed to obtain parameters useful to determine a pharmacokinetic profile. Often the samples are diluted, for example, using a buffer or pharmaceutically acceptable carrier as defined herein.
  • Pharmacokinetic standard curves are often generated using a chlorotoxin conjugate, serum and a pharmaceutical carrier as described herein.
  • the proportion of each chlorotoxin conjugate, concentrated source of sample (for example serum, urine, etc.) and pharmaceutical carrier often differs, for example, the concentration of compound of the present disclosure is often between about 10 ⁇ g/mL and about 4 ng/mL.
  • the standard curve is used to calculate the concentration of the compound in the sample.
  • pharmacokinetic parameters, or pharmacokinetic data are analyzed using standard pharmacokinetic data analysis methods, including concentration of chlorotoxin conjugates versus time.
  • a software program such as Phoenix WinNonlin 6.3 is used to analyze pharmacokinetics data.
  • the pharmacokinetic data analysis uses standard noncompartmental methods of intravenous bolus, intravenous infusion, or extravascular input as appropriate.
  • the pharmacokinetic data analysis uses nonstandard noncompartmental methods of intravenous bolus, intravenous infusion, or extravascular input as appropriate.
  • the data are analyzed by the mean serum concentration versus time. The data are also analyzed by individual subject followed by group summary statistics.
  • bioanalytical methods include the addition of chemicals to a sample containing a composition of which the pharmacokinetic profile is desired.
  • Addition of the chemical to the sample often comprises performing a chemical technique to measure the concentration of a composition or a metabolite thereof in a sample or, sometimes, in a biological matrix.
  • a chemical technique to measure the concentration of a composition or a metabolite thereof in a sample or, sometimes, in a biological matrix.
  • microscale thermophoresis, mass spectrometry often including liquid chromatorgraph and a triple quadropole mass spectrometer, tandem mass spectrometry, high sensitivity mass spectrometry for microdosing studeies and the like are often performed.
  • the disclosure further describes methods of administering compound and compositions of the present disclosure to a subject, often methods include intravenous administration of a chlorotoxin conjugate composition to a subject.
  • the method of administering a chlorotoxin polypeptide to a subject comprises intravenously administering a dose of from 0.8 to 25 mg of the chlorotoxin polypeptide to the subject, wherein the chlorotoxin polypeptide has at least 85% sequence identity with MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR; producing in the subject an average chlorotoxin polypeptide plasma area under the curve (average AUC) of from 50 to 120 per each 1 mg dosage of chlorotoxin polypeptide administered; and producing in the subject an average maximum chlorotoxin peptide blood plasma concentration (average C max ) of at least from 110 to 240 per each 1 mg dosage of chlorotoxin peptide administered.
  • the chlorotoxin polypeptide is conjugated to a fluorescent agent.
  • the disclosure further describes methods of treating and/or detecting cancer with chlorotoxin conjugate compositions following administration to a subject or contacting tumor tissue isolated from a subject, often the methods include intravenous administration of a chlorotoxin conjugate composition to a subject, in vivo contact of a tumor tissue or ex vivo contact of a tumor tissue from a subject.
  • the method treating and/or detecting cancer with chlorotoxin conjugate compositions following administration to a subject or contacting tumor tissue isolated from a subject often the methods include intravenous administration of a chlorotoxin conjugate composition to a subject, in vivo contact of a tumor tissue or ex vivo contact of a tumor tissue from a subject comprises intravenously administering a dose of from 0.8 to 25 mg of the chlorotoxin polypeptide to the subject, wherein the chlorotoxin polypeptide has at least 85% sequence identity with MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR; producing in the subject an average chlorotoxin polypeptide plasma area under the curve (average AUC) of from 50 to 120 per each 1 mg dosage of chlorotoxin polypeptide administered; and producing in the subject an average maximum chlorotoxin peptide blood plasma concentration (average C max ) of at least from 110 to 240 per each 1 mg dosage of chlorotoxin peptide administered.
  • average AUC
  • compositions of chlorotoxin conjugates often the chlorotoxin conjugate composition comprises a physiologically effective amount of a chlorotoxin conjugate, wherein the chlorotoxin conjugate comprises a chlorotoxin polypeptide having at least 85% sequence identity with MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR conjugated to a fluorescent dye, for example, as provided in the present disclosure, or a derivative thereof, wherein intravenous administration of the composition to a subject produces in the subject: an average chlorotoxin polypeptide plasma area under the curve (average AUC) of from 50 to 120 per each 1 mg dosage of chlorotoxin polypeptide administered; and an average maximum chlorotoxin peptide blood plasma concentration (average C max ) of at least from 110 to 240 per each 1 mg dosage of chlorotoxin peptide administered.
  • the chlorotoxin polypeptide is conjugated to a fluorescent agent.
  • the present disclosure provides, but is not limited to, methods for intraoperative imaging and resection of tumors with chlorotoxin conjugates detectable by fluorescence imaging that allows for intraoperative visualization of cancerous tissues, compositions that include the chlorotoxin conjugate, and methods for using the chlorotoxin conjugate.
  • the chlorotoxin is a targeting agent that directs the conjugate to a tissue of interest.
  • the chlorotoxin conjugate of the disclosure includes one or more labeling agents.
  • the labeling agent comprises a fluorescent moiety (e.g., red or near infrared emitting fluorescent moieties) covalently coupled to the chlorotoxin.
  • the labeling agent comprises a radionuclide.
  • tumors amenable to detection with a chlorotoxin conjugate of the present disclosure include, but are not limited to: adenocarcinoma, fibrosarcoma, hemangiosarcoma, mastocytoma, squamous cell carcinoma, chondrosarcoma, adenosquamous carcinoma, hemangiopericytoma, follicular carcinoma, meningioma, mucosal squamous cell cancer, glioma, sarcomas, such as soft-tissue sarcomas or the like.
  • Soft-tissue sarcomas amenable to detection with a chlorotoxin conjugate of the present disclosure include, but are not limited to: fat tissue tumors, liposarcomas, muscle tissue tumors including smooth muscle sarcomas and leiomyosarcomas, skeletal muscle sarcomas, rhabdomyosarcomas, peripheral nerve tumors, fibrous tissue tumors, myxofibrosarcomas, fibromatosis, joint tissue tumors, tumors of blood vessels and lymph vessels, angiosarcomas, tumors of peripheral nerves such as malignant peripheral nerve sheath tumors, malignant schwannomas, neurofibrosarcomas, fibrosarcomas, synovial sarcomas, malignant fibrous histiocytoma (MFH) hemangiosarcomas, lymphangiosarcomas, gastrointestinal stromal tumors, alveolar soft part sarcoma, dermatofibrosar
  • the chlorotoxin conjugates described herein can be used for detection and treatment of tumors present in any organ and in any anatomical location, including but not limited to, breast, lung, brain, colon, rectum, prostate, head, neck, stomach, anus, and/or vaginal tissues, for example.
  • Tumors of any grade or stage known to one of skill in the art, including low-grade tumors, are often detected by the chlorotoxin conjugates described herein.
  • tumor detection includes imaging, resection, diagnostics and treatment.
  • the present compounds are capable of passing across the blood brain barrier. Passing across the blood brain barrier is advantageous when detecting or treating a cancer cell in the brain, such as for example, a glioma cell or a brain tumor.
  • the dose of chlorotoxin conjugate is administered such that a threshold amount of chlorotoxin conjugate is achieved in the subject.
  • the threshold amount often depends upon the patient's age, weight, height, sex, general medical condition and previous medical history.
  • the threshold amount does not depend upon the patient's age, weight, height, sex, general medical condition and previous medical history.
  • dosage forms is devised by those skilled in the art, as shown, for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Edition (Lea & Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRC Press 1996).
  • compositions are often supplied as a kit comprising a container that comprises a chlorotoxin conjugate.
  • Chlorotoxin conjugates are often provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection.
  • a kit often includes a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a therapeutic conjugate.
  • Such a kit further comprises written information on indications and usage of the pharmaceutical composition.
  • Subjects include, but are not limited to humans, non-human primates, monkeys, cows, dogs, rabbits, pigs, guinea pigs, rats, mice and zebrafish.
  • a sample includes any sample isolated from a subject, for example but not limited to, blood, serum, plasma, circulating cells, urine, saliva, and/or tissue removed from the body such as in a biopsy. Samples are often prepared using methods known to those of ordinary skill in the art, for example, blood samples are collected and incubated at room temperature for about 0.5 hours up to 2 hours prior to centrifugation, serum removal and storage at at least about 20° C., but more often ⁇ 70° C.
  • the present disclosure provides methods for treating a disease or condition treatable by administering chlorotoxin.
  • the method includes administering an effective amount of a modified chlorotoxin peptide of the invention to a subject in need thereof.
  • an effective amount refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • Compositions containing such agents or compounds can be administered for prophylactic, enhancing, and/or therapeutic treatments.
  • An appropriate “effective” amount in any individual case may be determined using techniques, such as a dose escalation study.
  • the invention provides a method for treating a cancer that expresses chlorotoxin binding sites in a patient, comprising administering to a patient in need thereof an effective amount of a chlorotoxin variant of the invention.
  • the invention provides a method for treating a cancer that expresses chlorotoxin binding sites, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a chlorotoxin variant of the invention and a pharmaceutically acceptable carrier.
  • the invention provides a method for treating a tumor expressing chlorotoxin binding sites, comprising administering to a patient in need thereof an effective amount of a chlorotoxin variant of the invention.
  • the invention provides a method for inhibiting invasive activity of cells that express chlorotoxin binding sites, comprising administering an effective amount of a chlorotoxin variant to cells that express chlorotoxin binding sites.
  • the methods of treatment of the invention are applicable to human and animal subjects in need of such treatment.
  • malignant cancers include gliomas, astrocytomas, medulloblastomas, choroid plexus carcinomas, ependymomas, meningioma, glioblastoma, ganglioma, pheochromocytoma, and metastatic brain tumors, other brain tumors, neuroblastoma, head and neck cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, intestinal cancer, pancreatic cancer, colon cancer, liver cancer, kidney cancer, skin cancer, sarcomas (over 30 types), osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, carcinomas, melanomas, ovarian cancer, cervical cancer, lymphoma, thyroid cancer, anal cancer, colo-rectal cancer, endometrial cancer, germ cell tumors, lary
  • the chlorotoxin conjugate is administered to an individual having or suspected of having a tumor, such that the conjugate binds specifically to the tumor.
  • Such methods are useful in reducing the likelihood that the individual will develop a tumor, that one or more tumors in the individual will increase in size, that one or more tumors in the individual will metastasize, and/or that the cancer will progress by some other measure.
  • the term “metastasis” refers to the spread of tumor cells from one organ or tissue to another location, and also refers to tumor tissue that forms in a new location as a result of metastasis.
  • the chlorotoxin conjugate is useful for the treatment and/or diagnosis of neuroectodermal tumors such as gliomas, medulloblastomas, neuroblastomas, pheochromocytomas, melanomas, peripheral primitive neuroectodermal tumors, small cell carcinoma of the lung, Ewing's sarcoma, and metastatic tumors in the brain.
  • neuroectodermal tumors such as gliomas, medulloblastomas, neuroblastomas, pheochromocytomas, melanomas, peripheral primitive neuroectodermal tumors, small cell carcinoma of the lung, Ewing's sarcoma, and metastatic tumors in the brain.
  • the chlorotoxin conjugate is useful for the treatment and/or diagnosis of brain tumors, including but not limited to, glioma, including glioblastoma multiforme, anaplastic astrocytomas, low grade gliomas, pliocytic astrocytomas, oligodendrogliomas, gangliomas, meningiomas, and ependymomas.
  • glioma including glioblastoma multiforme, anaplastic astrocytomas, low grade gliomas, pliocytic astrocytomas, oligodendrogliomas, gangliomas, meningiomas, and ependymomas.
  • the compounds of the present disclosure are used to detect and/or treat soft-tissue sarcomas.
  • Soft-tissue sarcomas are a group of malignant tumors that form in fat, muscles, nerves, joints, and blood vessels. In 2012, it was estimated that approximately 11,280 Americans would be diagnosed with soft-tissue sarcomas and approximately 3,900 would be expected to die from soft-tissue sarcomas. Soft tissue and bone sarcoma incidence rates have increased slightly over the past 30 years; however, soft-tissue sarcoma is more deadly, possibly because the lack of specific symptoms at early disease stages may lead to delays in diagnosis.
  • the present invention provides such methods of detection, imaging, visualization, analysis and treatment for these and other uses that should be apparent to those skilled in the art from the teachings herein.
  • the present invention is based in part upon the identification by the inventors that soft-tissue sarcomas have a high level of uptake of the conjugate compared to other tumors or normal tissues and are particularly well-suited for detection by means of administering a chlorotoxin conjugated to a labeling agent for detection, visualization, imaging, or analysis.
  • Such visualization can be during or related to surgical (intraoperative) resection or during or related to initial identification of the sarcoma or during or related to monitoring of the sarcoma relevant to treatment.
  • Real-time intraoperative visualization of solid tumors enables more complete resection while sparing surrounding normal tissue. Improvement in intraoperative tumor visualization would be of benefit for any resectable solid tumor, as it would enable surgeons to better determine the extent of local invasion as well as the presence of metastatic spread in nearby lymph nodes and fatty tissue. This kind of information would be helpful in making surgical decisions, for example, decisions regarding which patients would respond well to limb-sparing approaches in the treatment of sarcomas.
  • the present invention shows that soft-tissue sarcomas are particularly identifiable when bound by a chlorotoxin conjugate, which can be effectively used to detect these sarcomas, especially during or related to surgery and intraoperative resection.
  • the chlorotoxin conjugate can be used alone or on combination with other detection agents, to detect, image, visualize, or analyze the tumor in advance of, during, or following anti-tumor treatments, which can include surgery and surgical resection, chemotherapy, radiation therapy, and immunotherapy.
  • the chlorotoxin conjugate can be used alone or with other detection agents for follow-up monitoring post treatment as well as for general monitoring for full-body screening.
  • Low-grade tumors generally tend to be slow growing, slower to spread, and often have better prognosis than higher-grade tumors, making them more curable with surgical resection than high-grade tumors, which may need more systemic treatment.
  • the inventors show that use of a chlorotoxin conjugated to a labeling agent can be particularly effective in detection, imaging, visualization, or analysis of low-grade tumors, such as meningiomas, allowing their complete resection before they metastasize or spread.
  • Intraoperative resection of tumor types may vary depending on the anatomic location and type of tumor. For example, when a tumor is located in brain tissue, the surgeon is likely to require perfect or near perfect specificity between a tumor imaging or detection agent and the tumor tissue so that only diseased tissue is resected. On the other hand, when the tumor is located in a tissue where wider margins are generally resected, such as breast or mammary cancer or colon cancer, or in cancers where the tumor is likely to spread locally, such as squamous cell carcinoma, it would be advantageous for a surgeon to be able to use a tumor imaging or detection agent to identify peritumoral tissue that is likely to become tumor tissue.
  • Soft-tissue sarcomas can develop from soft tissues like fat, muscle, nerves, fibrous tissues, blood vessels, or deep skin tissues. They can be found in any part of the body. Most of them develop in the arms or legs. They can also be found in the trunk, head and neck area, internal organs, and the area in back of the abdominal cavity (known as the retroperitoneum).
  • Soft-tissue sarcomas that may be amenable to detection with a chlorotoxin conjugate of the present invention include, but are not limited to: fat tissue tumors, muscle tissue tumors, skeletal muscle sarcomas, rhabdomyosarcomas, peripheral nerve tumors, fibrous tissue tumors, myxofibrosarcomas, fibromatosis, joint tissue tumors, tumors of blood vessels and lymph vessels, angiosarcomas, gastrointestinal stromal tumors, alveolar soft part sarcoma, dermatofibrosarcoma protuberans (DFSP), desmoplastic small round cell tumour, epithelioid sarcoma, extra skeletal myxoid chondrosarcoma, and giant cell fibroblastoma (GCF).
  • fat tissue tumors e.g., muscle tissue tumors, skeletal muscle sarcomas, rhabdomyosarcomas, peripheral nerve tumors, fibrous tissue tumors
  • Liposarcomas that start in the body's fat cells are called liposarcomas. They can grow anywhere in the body and most commonly affect people aged 50-65 years. Some grow very slowly, taking many years to develop, whereas others grow more quickly.
  • Muscle tissue sarcomas include smooth muscle sarcomas and skeletal muscle sarcomas. Smooth muscle forms the walls of internal organs such as the stomach, intestine, womb (uterus), and blood vessels. The muscle causes these organs to contract, which happens without our control. Smooth muscle is also called involuntary muscle. Sarcomas that develop in smooth muscle are called leiomyosarcomas. They are one of the more common types of sarcoma and can occur anywhere in the body, especially in the back of the abdominal area (retroperitoneum). Leiomyosarcomas are less often found in the deep, soft tissues of the legs or arms. They tend to occur in adults, particularly in the elderly. Skeletal muscles are the active muscles in our arms and legs or other parts of the body that we control. They are voluntary muscles and sometimes called striated muscles because the cells look stripy when examined under a microscope.
  • rhabdomyosarcomas Sarcomas that grow in the voluntary muscles of the body are called rhabdomyosarcomas. They are found mostly in the head and neck, but also in organs such as the bladder, vagina and the arms or legs. Rhabdomyosarcomas are more commonly diagnosed in children than in adults.
  • Peripheral nerve tumors can be found in the peripheral nervous system, which consists of all the nerves that run throughout the body. Sarcomas of the peripheral nerves develop in the cells that cover the nerves. They're known as malignant peripheral nerve sheath tumors (MPNST) and can occur anywhere in the body. There are different types of MPNSTs, including malignant schwannomas and neurofibrosarcomas. They most commonly occur in people who have a rare genetic disorder called neurofibromatosis (von Recklinghausen's disease).
  • Fibrous tissue tumors occur in tissues that join muscles to bones. This tissue is made up of cells called fibrocytes. A sarcoma of the fibrous tissue is called a fibrosarcoma. They are most commonly found on the arms, legs or trunk, but can occur deeper in the body. They can occur at any age but are more commonly seen in people aged 20-60 years. Most people first notice them as a painless, firm lump.
  • synovial sarcomas Soft-tissue sarcomas that develop very close to the body's joints are known as synovial sarcomas. They commonly develop near, but not inside, joints such as the knee or elbow, but they can occur in any part of the body. They usually appear as hard lumps and are more common in children and young adults.
  • Blood and lymph vessel tumors include sarcomas that start from the cells that make up the walls of blood or lymph vessels and are called angiosarcomas. Haemangiosarcomas develop from blood vessels and lymphangiosarcomas develop from the lymph vessels.
  • Angiosarcomas are sarcomas that sometimes occur in a part of the body that has been treated with radiotherapy many years before.
  • Gastrointestinal stromal tumors are soft-tissue sarcomas that develop in nerve cells in the walls of the digestive system.
  • low-grade tumors can be detected with a chlorotoxin conjugated to a labeling agent. This is particularly useful since low-grade tumors have a better prognosis if they can be fully resected.
  • the invention is based in part on the identification by the inventors of an optimal dose for tumor imaging in dogs of at least about 0.8 mg/m 2 .
  • dosage for the chlorotoxin conjugate will be determined based on the amount of conjugate administered and the amount of time after administration after which the imaging is performed.
  • the optimal dose for tumor imaging is in the range of about 0.85 mg/m 2 to about 1.2 mg/m 2 , or in the range of about 0.9 to about 1.1 mg/m 2 . At doses up to 0.9 mg/m 2 , signal in gross tumor samples increases as a function of dose.
  • the amount of chlorotoxin conjugate that can be administered can be in the range of about 1.3 mg/m 2 to about 2.5 mg/m 2 , or in the range of about 2.6 mg/m 2 to about 3.5 mg/m 2 , or in the range of about 3.6 mg/m 2 to about 4.5 mg/m 2 , or in the range of about 4.6 mg/m 2 to about 5.5 mg/m 2 , or upwards of 5.5 mg/m 2 .
  • the invention provides a method for imaging a tissue imagable by chlorotoxin.
  • a tissue imagable by chlorotoxin is contacted with a chlorotoxin conjugate.
  • the imaging method is a fluorescence imaging method. Representative methods for making and using fluorescent chlorotoxin conjugates are described in U.S. Patent Application Publication No. 20080279780 A1, Fluorescent Chlorotoxin Conjugate and Method for Intra-Operative Visualization of Cancer, and in U.S. Patent Application Publication No. 20130195760, Chlorotoxin Variants, Conjugates, And Methods For Their Use, both of which are expressly incorporated herein by reference in their entirety.
  • chlorotoxin conjugates can be administered to human and animal subjects, such as with a pharmaceutically acceptable carrier.
  • the composition includes a pharmacologically effective amount of a modified chlorotoxin conjugate.
  • An effective amount can be routinely determined by established procedures.
  • An effective amount is an amount sufficient to occupy chlorotoxin binding sites in cancer cells, but low enough to minimize non-specific binding to non-neoplastic tissues.
  • An effective amount optimizes signal-to-noise ratio for intra-operative imaging.
  • the disclosure provides methods for detecting a tissue using the chlorotoxin conjugates.
  • the chlorotoxin conjugates of the invention target and are bound by chlorotoxin binding sites. It will be appreciated that chlorotoxin binding sites may take two forms: sites that bind chlorotoxin and sites that bind the chlorotoxin conjugates of the invention. It will be appreciated that chlorotoxin binding sites may be distinct from chlorotoxin conjugate binding sites.
  • a method for differentiating foci of cancers that express chlorotoxin binding sites from non-neoplastic tissue includes contacting a tissue of interest with a chlorotoxin conjugate having affinity and specificity for cells that express chlorotoxin binding sites, wherein the chlorotoxin conjugate comprises one or more red or near infrared emitting fluorescent moieties covalently coupled to a chlorotoxin, and measuring the level of binding of the chlorotoxin conjugate, wherein an elevated level of binding, relative to normal tissue, is indicative that the tissue is neoplastic.
  • a method for detecting cancers that express chlorotoxin binding sites includes the steps of contacting a tissue of interest with a chlorotoxin conjugate having affinity and specificity for cells that express chlorotoxin binding sites, wherein the chlorotoxin conjugate comprises one or more red or near infrared emitting fluorescent moieties covalently coupled to a chlorotoxin, and measuring the level of binding of the chlorotoxin conjugate, wherein an elevated level of binding, relative to normal tissue, is indicative that the tissue is neoplastic.
  • a method for determining the location of cancer cells that express chlorotoxin binding sites in a patient intra-operatively includes the steps of administering a pharmaceutical composition to a patient, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an amount of a chlorotoxin conjugate sufficient to image cancer cells that express chlorotoxin binding sites in vivo, wherein the chlorotoxin conjugate comprises one or more red or near infrared emitting fluorescent moieties covalently coupled to a chlorotoxin, measuring the level of binding of the chlorotoxin conjugate by fluorescence imaging to determine the location of cancer cells that express chlorotoxin binding sites, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of cancer cells that express chlorotoxin binding sites; and surgically removing from the patient at least some cells that express chlorotoxin binding sites located by fluorescence imaging.
  • Imaging methods for detection of cancer foci disclosed herein are applicable to mouse and other animal models of cancer as well as to veterinary practice.
  • the present invention provides methods for intraoperative imaging and resection of tumors with a chlorotoxin conjugates detectable by fluorescence imaging that allows for intraoperative visualization of cancerous tissues, compositions that include the chlorotoxin conjugate, and methods for using the chlorotoxin conjugate.
  • the chlorotoxin is a targeting agent that directs the conjugate to a tissue of interest.
  • the chlorotoxin conjugate of the invention includes one or more labeling agents.
  • the labeling agent comprises a fluorescent moiety (e.g., red or near infrared emitting fluorescent moieties) covalently coupled to the chlorotoxin.
  • the labeling agent comprises a radionuclide.
  • red or near infrared emitting fluorescent moiety refers to a fluorescent moiety having a fluorescence emission maximum greater than about 600 nm.
  • the fluorescent moieties are derived from fluorescent compounds characterized by emission wavelength maxima greater than about 600 nm to avoid autofluorescence, emission that travels through millimeters to one centimeter of tissue/blood/fluids, emission that is not absorbed by hemoglobin, other blood components, or proteins in human or animal tissue.
  • the emission wavelength maximum is greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, or greater than 950 nm.
  • the fluorescent moiety is covalently coupled to the chlorotoxin to allow for the visualization of the conjugate by fluorescence imaging.
  • the fluorescent moiety is derived from a fluorescent compound. Suitable fluorescent compounds are those that can be covalently coupled to a chlorotoxin without substantially adversely affecting the targeting and binding function of the chlorotoxin conjugate. Similarly, suitable fluorescent compounds retain their fluorescent properties after conjugation to the chlorotoxin.
  • the dosage of administered chlorotoxin conjugates may vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of chlorotoxin conjugated to a chemotherapeutic, an anti-cancer agent, or an anti-cancer drug that is effective to achieve inhibition, shrinkage, killing, minimization, or prevention of metastasis. In many cases, it is desirable to provide the recipient with a dosage of a chlorotoxin conjugate that is in the range of from about 3 mg to about 6 mg, although a lower or higher dosage also may be administered as circumstances dictate.
  • Administration of a chlorotoxin conjugate to a subject can be topical, inhalant, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection.
  • the administration may be by continuous infusion or by single or multiple boluses.
  • Additional routes of administration include oral, mucosal-membrane, pulmonary, and transcutaneous.
  • Oral delivery is suitable for polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid-based systems (see, for example, DiBase and Morrel, “Oral Delivery of Microencapsulated Proteins,” in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)).
  • the feasibility of an intranasal delivery is exemplified by such a mode of insulin administration (see, for example, Hinchcliffe and Ilium, Adv. Drug Deliv. Rev. 35:199 (1999)).
  • Dry or liquid particles comprising a chlorotoxin conjugate can be prepared and inhaled with the aid of dry-powder dispersers, liquid aerosol generators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35:235 (1999)).
  • dry-powder dispersers liquid aerosol generators
  • nebulizers e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35:235 (1999)
  • AERX diabetes management system which is a hand-held electronic inhaler that delivers aerosolized insulin into the lungs.
  • Transdermal delivery using electroporation provides another means to administer a chlorotoxin conjugate.
  • a pharmaceutical composition comprising a chlorotoxin conjugate can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the conjugate is combined with a pharmaceutically acceptable carrier.
  • a composition is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • Other suitable carriers are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • a pharmaceutical composition comprising a chlorotoxin conjugate can be furnished in liquid form, in an aerosol, or in solid form.
  • Liquid forms are illustrated by injectable solutions, aerosols, droplets, topological solutions and oral suspensions.
  • Exemplary solid forms include capsules, tablets, and controlled-release forms. The latter form is illustrated by miniosmotic pumps and implants (Bremer et al., Pharm. Biotechnol.
  • dosage forms can be devised by those skilled in the art, as shown, for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5.sup.th Edition (Lea & Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences, 19.sup.th Edition (Mack Publishing Company 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRC Press 1996).
  • compositions may be supplied as a kit comprising a container that comprises a chlorotoxin conjugate.
  • Therapeutic conjugates can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection.
  • a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a therapeutic conjugate.
  • Such a kit may further comprise written information on indications and usage of the pharmaceutical composition.
  • a method describing steps (a), (b), and (c) can be performed with step (a) first, followed by step (b), and then step (c).
  • the method can be performed in a different order such as, for example, with step (b) first followed by step (c) and then step (a).
  • steps can be performed simultaneously or separately unless otherwise specified with particularity.
  • This example demonstrates the production and stability of Compound 16 under various pH and temperature storage conditions over time.
  • the peptide portion of Compound 16 is a targeting peptide (modified chlorotoxin) conjugated to the fluorescent dye.
  • the targeting peptide binds selectively to cancerous cells and the dye portion facilitates detection via imaging.
  • the peptide is a 36 amino acid modified chlorotoxin (having a sequence of H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Arg-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH) wherein two of three lysine amino acids in native chlorotoxin are substituted with arginine (K115R and K23R) to facilitate the subsequent conjugation with a fluorophore to the
  • Ammonium Bicarbonate Purification The reaction solution was filtered and then diluted with 400 mL of water. The solution was loaded on an RP-HPLC column equilibrated with 0.1M ammonium bicarbonate, and the product recovered by applying a linear gradient of acetonitrile.
  • the purity, estimated concentration, and estimated amount of product in each fraction are reported in Table 15.
  • the estimation was based on the peak area observed during release analysis of a solution of the product at a concentration of 1 mg/mL in water.
  • the main pool (fractions 6-1-5 to 6-1-8, purity 97%) was combined and transferred to the salt exchange step. Samples of the main pool were analyzed for a period of 5 day of storage protected from light at ⁇ 8° C. The material was stable under these conditions.
  • Ammonium Acetate Salt Exchange The primary purification main pool solution ( ⁇ 200 mL) was diluted with 100 mL of water and loaded on an RP-HPLC column equilibrated with 0.1M ammonium bicarbonate. Following loading of the sample, the column was equilibrated with 4 bed volumes of 0.1M ammonium acetate adjusted to pH 7.6 with ammonium hydroxide. Finally the column was equilibrated with 0.01M ammonium acetate pH 7.6 and the product was recovered by applying a linear gradient of 0.01M ammonium acetate pH 7.6 in 75% Acetonitrile.
  • the product eluted as a single peak at approximately 39% acetonitrile concentration.
  • the fractions were collected and analyzed by analytical HPLC. During analysis, the fractions were stored at ⁇ 8° C. protected from light.
  • the purity, estimated concentration, and estimated amount of product in each fraction are reported in Table 16.
  • the estimation was based on the peak area observed during release analysis of a solution of the product at a concentration of 1 mg/mL in water.
  • the main pool (fractions 7-1-2 to 7-1-6, purity 97.2%) were combined and transferred to lyophilization. Samples of the main pool were analyzed for a period of 5 day of storage protected from light at ⁇ 8° C. The material was stable under these conditions. Dilution with a volume equal to approximately half the main pool volume provided a stable solution without the presence of a precipitate. This dilution was utilized at the preparative scale.
  • Lyophilization The main pool at stage 7 was diluted with 100 mL of water and lyophilized over a period of 4 days using a bottle lyophilizer. There was no problem with solubility after dilution as had been observed with the ammonium bicarbonate main pool; a sample was diluted to 15% acetonitrile without any observed precipitation. During lyophilization, the product formed a stable self-supporting cake.
  • Reconstitution The product was readily soluble in water at 1, 5 and 10 mg/mL. Water was selected as the reconstitution solution. Additionally, an LC-MS analysis was performed on the final material.
  • Compound 16 was stable for up to 14 days at pH 7.5 and 8.5 when stored in the dark at 4° C. in 10 mM Tris, 5% Dextrose and is sensitive to low pH and temperatures above 4° C.
  • This example shows the stability of Compound 16 with respect to pH and provides an evaluation of the use of alternative buffers and various excipients and configurations.
  • This example further shows that various classes of excipients protect Compound 16 from thermal, photo, oxidative and freeze/thaw stress.
  • formulations were stored in microcentrifuge tubes with an air atmosphere and assayed after 2 d and 5 d at 40° C. in the dark, after 3 d at room temperature (rt) in the dark (dk) or in ambient light (lt), and after 3 ⁇ freeze thaw (F/T) between ⁇ 20° C. and room temperature in the dark.
  • Formulations were evaluated by visual examination, centrifugation, RP-HPLC, concentration by A 786 , pH, and SDS-PAGE.
  • the timepoints were 4, 7, and 14 days at room temperature with evaluation by visual examination, centrifugation, RP-HPLC, concentration by A 786 , pH, and SDS-PAGE. Where extra material remained, additional stressing conditions were evaluated for further information.
  • the formulations were contained in microcentrifugation tubes in an air environment.
  • Dialysis buffers were prepared immediately before use by combining a stock from Table 19 with sparged water (sparged by bubbling with argon for 15 min-3 h).
  • Compound 16 was prepared at 3 mg/mL in water (see concentration by A 786 below). Slide-a-lyzer cassettes were pre-soaked in water, then the Compound 16 was dispensed into the cassettes ( ⁇ 11 mL for formulation 1, ⁇ 5 mL for formulations 2-6, ⁇ 2.7 mL for formulations 7-12). The cassettes were placed into beakers containing 550 mL of the corresponding formulation buffer. Samples were formulated by dialyzing with stirring in the dark at room temperature (on a multiposition stir plate, covered with a box covered with aluminum foil). Dialysis buffer was changed after 1 h, again after 1.25 h, and allowed to continue overnight.
  • the formulation was placed in a fresh tube and combined with additional sterile 0.25M or 0.5M buffer stock to yield a new formulation with 30 mM concentration of buffer. All formulations were then dispensed into 1.5 mL microcentrifuge tubes, 0.5 mL per tube.
  • the designed stability timepoints were 4, 7, and 14 days at room temperature. Where extra material remained, additional testing was performed. One tube of each formulation was assayed immediately (“t0”). For additional information, remaining material from these t0 tubes was then frozen at ⁇ 20° C., then thawed at 5° C., and assayed for pH and concentration (“1 ⁇ F/T”). Other tubes were incubated in the dark at room temperature (approximately 22-23° C.). A tube was removed and assayed at 4, 7, and 14 d room temperature (rt) (“4 d rt”, “7 d rt”, “14 d rt”).
  • Samples were examined visually for clarity, color, and visible particulates. They were then mixed, withdrawn into glass Pasteur pipets, and examined again. Samples were centrifuged ⁇ 10,000 rpm for ⁇ 2 min. Samples were then examined visually for any visible pellet.
  • 100 ⁇ l of sample was placed in an HPLC vial insert, placed in an amber HPLC vial, and held at 2-8° C. before the 2 ⁇ l injection.
  • the formulation buffer was injected twice at the beginning of each run, and a matched formulation buffer blank was run each time before injection of a sample in a new buffer.
  • the spectrophotometer was blanked with matching formulation buffer. Concentration was calculated using the extinction coefficient. The pH was measured using a calibrated micro pH electrode.
  • Samples were prepared per the manufacturer's recommendations and then heated at 70° C. for 10 min. 2 ⁇ g in 10 ⁇ L was loaded onto the SDS-PAGE gels and then run at 200V for approximately 35 min. After electrophoresis, the gels were washed in water for 5 min at room temperature, 3 times. Then they were stained at room temperature for 1.5 h, destained in water overnight, and then destained again with water and imaged on the same day.
  • Formulations were produced by dialyzing a stock of Compound 16 at 3 mg/mL in water into various buffers. The formulations were then dispensed into microcentrifuge tubes, in air, and stored in the dark at room temperature for up to 14 days. The results from visual examination of the formulations are given in Table 21.
  • FIG. 1 shows SDS-PAGE analysis of the formulations after 14 days at room temperature.
  • FIGS. 1A and 1B show SDS-PAGE analysis of, from left to right, (molecular weight marker) MWM, 1, 2, 2B, 4, 6, 6B, 9, 10 and reference.
  • FIG. 1A was performed with a reducing agent and
  • FIG. 1B was performed without a reducing agent.
  • FIG. 1C shows SDS-PAGE analysis of, from left to right, 7, 8, 11, 12, reference and MWM (no reducing agent).
  • Reference Pilot Lot, Sublot #2.
  • MWM 188 k, 98 k, 62 k, 49 k, 38 k, 28 k, 17 k, 14 k, 6 k, 3 k. Arrow points to a higher molecular weight species.
  • Compound 16 was formulated by additional means. Stocks were prepared and the pH of some stocks was adjusted to near 6.8 to avoid pH shift upon addition to formulations (Table 25). All stocks were 0.2 ⁇ m filtered except BHT, BHA, propyl gallate, and polysorbates. Polysorbate and Nal stocks were stored with an argon blanket. His, Met, Nal, polysorbate, BHT, BHA, and propyl gallate stocks were stored in the dark.
  • Compound 16 was prepared at 10 mg/mL in argon-sparged water.
  • the Compound 16 stock was combined with buffer stock, osmolyte stock, and water to make parent stocks A-E containing Compound 16 (Table 26).
  • the pH of each soluble parent stock was adjusted (from starting values of 6.6-6.7) to 6.8 with 0.1N NaOH.
  • One of the 0.5 mL tubes was placed in the dark at room temperature and the other 0.5 mL tube was placed in the light at room temperature.
  • the tubes were placed on their side and exposed to ambient light.
  • a volume of 0.5 mL was chosen for light exposure, as previous work had shown 0.3 mL samples are prone to precipitation and degradation after light exposure in this configuration.
  • Stability samples were assayed by visual inspection, centrifugation, RP-HPLC, A 786 , pH, and, in some cases, SDS-PAGE. Samples were held in the dark at 5° C. when not in use. Samples were examined visually for clarity, color, and visible particulates. Samples were inverted three times and examined again. Samples were gently mixed, then centrifuged ⁇ 10,000 rpm for ⁇ 2 min. Samples were examined visually for a pellet. Remaining assays were performed on the supernatant.
  • Samples were prepared per the manufacturer's recommendations and then heated at 70° C. for 10 min. 2.5 ⁇ g in 10 ⁇ L was loaded onto the SDS-PAGE gels and then run at 165-200V. After electrophoresis, the gels were washed in water for 5 min at room temperature, 3 times. Gels were stained at room temperature for 1 h, destained in water overnight, and then destained again with water and imaged on the same day.
  • Formulations were produced by dissolving Compound 16 at 10 mg/mL in water, diluting into the buffer and osmolyte, adjusting the pH, adding final additives and water as needed (Table 27).
  • Formulations #6 140 mM NaCl
  • #12 (1 mM EDTA)
  • #18 (0.2% Nal) had immediate gross precipitation and were discarded.
  • the material was clear, green, and had no significant visible particle formation at all timepoints tested (Table 28).
  • FIGS. 2A and 2B show SDS-PAGE analysis of, from left to right, (molecular weight marker) MWM, 3, 4, 5, 8, 9, 10, 11, 13, 14, 15, 16, 17, 19, reference.
  • FIG. 2A was performed with a reducing agent and FIG. 2B was performed without a reducing agent.
  • 2C shows SDS-PAGE analysis of, from left to right, 1 t0, 3 t0, 5 t0, 1, 2, 3, reference, MWM, 1 t0, 3 t0, 5 t0, 1, 2, 3, reference.
  • lanes to the left of MWM were performed with a reducing agent and lanes to the right of MWM were performed without a reducing agent.
  • Reference Pilot Lot, Sublot #2 ( ⁇ 20° C.).
  • MWM top to bottom: 188 k, 98 k, 62 k, 49 k, 38 k, 28 k, 17 k, 14 k, 6 k, 3 k.
  • Black arrows point to higher molecular weight species, green arrow points to new band in formulation 19, and red arrow points to what may be reduced material in formulations 5 and 15.
  • FIGS. 2A and 2B show SDS-PAGE analysis of, from left to right, (molecular weight marker) MWM, reference, 3, 4, 5, 8, 9, 10, 11, 13, 14, 15, 16, 17, 19.
  • FIG. 3A was performed with a reducing agent and
  • FIG. 3B was performed without a reducing agent.
  • FIG. 3C shows SDS-PAGE analysis of, from left to right, 3 F/T, 13 F/T, 14 F/T, 1, 2, 3, reference, MWM, 3 F/T, 13 F/T, 14 F/T, 1, 2, 3, reference.
  • Formulations were stored in microcentrifuge tubes with an air atmosphere and assayed after 2 days and 5 days at 40° C. in the dark, after 3 days at room temperature (rt) in the dark or in ambient light, and after 3 ⁇ freeze/thaw between ⁇ 20° C. and room temperature in the dark.
  • This example describes the stability of Compound 16 in different vial types and in different atmospheric (headspace) environments.
  • KNTi-0303 the active pharmaceutical ingredient of BLZ-100
  • amber glass, clear glass, CZ, and Type 1+ glass vials with air or N 2 headspace The purity of samples was assessed by RP-HPLC, as shown in Table 31. All samples degraded significantly upon incubation at 40° C. In some cases, the purity values after 8 d at 40° C. were similar or even higher than the values after 5 d at 40° C. Both the 5 d and 8 d samples were reanalyzed together the following week, and the purity values as well as chromatogram shapes were comparable to the original analyses.
  • the 2 d, 5 d, and 8 d vials were all in the 40° C. incubator on the same days, with the vials being removed after the set number of days, placed at 5° C. in the dark, and then analyzed within 1 d.
  • This example shows various lyophilization conditions for Compound 16.
  • Lyophilized Compound 16 was reconstituted by addition of 1.2 mL of water and followed with gentle mixing for ⁇ 1 min.
  • the reconstituted Compound 16 was a clear, emerald green solution with no visible particles and no pellet observed after centrifugation (15,000 ⁇ g, 5 min).
  • the formulation volumes were each 1.2 mL and lyophilized formulations were reconstituted with 1.1 mL of water followed by gentle mixing for ⁇ 1 min. No visible aggregates were observed during or after reconstitution.
  • Reconstituted samples were analyzed for oxidation/purity by RP-HPLC, mass determined by RP-HPLC and/or OD786, aggregation measured by pellet formation after centrifugation, MFI and/or SDS-PAGE, charge heterogeneity determined by cIEF, secondary structure indicated by FTIR, pH measurements as described herein (the pH ranged from 6.8-7.0) and the KF titration determined (residual moisture was ⁇ 0.5%).
  • This example shows the performance of ICG in targeting tumor tissue compared to normal tissue, often as the signal/noise ratio and the biodistribution of ICG conjugates 24 hours after injection into a subject.
  • N-terminal PEGylation of chlorotoxin (CTX) and Compound 76 prior to conjugation with ICG minimizes product heterogeneity resulting from potential ICG conjugation to the N-terminus in addition to the K27 site (the peptide sequence of Compound 76 having a sequence of H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Ala-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Ala-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH).
  • PEGylation often improves solubility in aqueous solutions and the signal to noise ratio during imaging of cancerous tissue.
  • Compound 76 Three derivatives of Compound 76 were prepared, i.e., PEGaldehyde derivatives of 2 kD, 5 kD, and 40 kD. These were obtained by PEGylating Compound 76 using 2 kD, 5 kD and 40 kD PEGaldehyde derivatives, respectively. 5 kD-PEGylated modified chlorotoxin (Compound 76-5 kD) was obtained by PEGylating Compound 76 using a 5 kD polydisperse PEGaldehyde derivative.
  • mice bearing U87 flank xenografts were injected with 2 nmol of each chlorotoxin based conjugate through the tail vein (IV). Twenty four hours after injection, the mice were euthanized and the tumor and leg muscle were resected or the brain, heart, liver, kidney, spleen, and blood were collected. Tumor and leg muscle resected tissue was imaged with the IVIS Spectrum equipped with a 745 nm excitation filter and an 820 nm emission filter. All tissue was frozen in OCT and stored.
  • ROI's regions of interest
  • Representative fluorescent images from each of the conjugates are shown ( FIG. 8 ).
  • the capsule surrounding the tumor tissue has a high level of signal compared to the tumor cells ( FIG. 9 ).
  • Compound 76-2 kD, Compound 76-5 kD, Compound 76-40 kD targeted the tumor tissue.
  • the free dye is not detected in the tumor 24 hours after injection.
  • PEGylated chlorotoxin conjugated to the fluorophore ICG binds to tumor tissue and is detected 24 h after injection. Tumor binding is specific to the properties of PEGylated chlorotoxin as the free dye was not detectable in tumors after 24 h.
  • Biodistribution patterns 24 h after injection show that Compound 76-5 kD distributes mainly to the liver and kidney with a low signal detected in the spleen and heart. Little or no signal was detected in normal brain ( FIG. 10 ). Additionally, significant signal was not detected in the great vessels of the heart, which has been observed with the IR800 and Cy5.5 conjugates in past experiments ( FIG. 11 ).
  • ICG free dye is not detectable in liver, kidney, spleen, or brain. Free dye is detectable in the fecal matter indicating that it is cleared from the blood by the liver in into the bile.
  • the biodistribution patterns of the conjugates therefore more closely resembles that which is seen for other CTX conjugates while the free dye biodistribution is similar to what is reported for non-conjugated ICG.
  • This example shows the use of Compounds 1-720 in targeting tumor tissue compared to normal tissue, as the signal/noise ratio and the biodistribution of chlorotoxin conjugate compounds 1 to 720 at 24 hours after injection into a subject.
  • Compounds 1-720 preferentially target and label tumor tissue and capsules surrounding tumors. Biodistribution patterns 24 h after injection show that Compounds 1-720 distribute mainly to the liver and kidney with a low signal detected in the spleen and heart. Little or no signal is detected in normal brain or in great vessels of the heart.
  • This example shows the optimal imaging time and dose of Compound 16 in mice and further shows that Compound 16 targets U87 human glioma cells implanted into the brains of mice.
  • Compound 16 signal in tumor compared to normal brain was compared to the SNR calculated in subcutaneous U87 flank xenografts using both whole tissue imaging and sliced tissue analysis.
  • mice bearing U87 human glioma cell line cultured using standard culture conditions in DMEM (Invitrogen), 10% Fetal Bovine Serum (Qualified #26140, Invitrogen), Pen/Strep (Invitrogen) xenografts in the flank were injected through the tail vein with 0.6, 2, 6, 10, or 20 nmol of Compound 16 in a total volume of 100 ⁇ l.
  • Mice were euthanized 1, 2, or 3 days after injection. Tumor, muscle, and skin were collected for all mice. Brain, heart, liver, and kidney were dissected for a subset of mice. The tissues were imaged using the IVIS Spectrum (Perkin Elmer) and quantified using Living Image software (Perkin Elmer).
  • the tumor tissue was frozen on dry ice in OCT, sliced into 12 ⁇ m sections, and scanned on the Odyssey CLx near-infrared imaging system (Li-Cor Biosciences) using the 800 nm channel (785 nm excitation). The tissue was scanned using the “auto” intensity setting and 21 ⁇ m resolution. Images were analyzed using the Image Studio software (Li-Cor) by measuring the fluorescent signal within a region of interest (ROI) drawn in each tissue image.
  • Orthotopic xenograft implants were generated using five week old female Nu/Nu mice (Harlan Laboratories) anesthetized with isoflurane. The scalp was swabbed with providone-iodine and alcohol. Using a scalpel, an incision was made in the scalp down the midline in the area of the cerebral cortex. A burr hole was drilled through the skull using a micro-drill fitted with a 0.9 mm bit. The burr hole was placed into the right cerebral hemisphere approximately 1 mm lateral (right) of the sagittal suture, 2 mm anterior to the lambdoid suture, and 2 mm deep.
  • mice with orthotopic U87 brain tumors and three mice with U87 flank tumors received tail vein injections of 100 ⁇ l of a 10 nmol/100 ⁇ l dose of Compound 16.
  • the brains bearing orthotopic xenografts, flank tumors, and normal brain were excised and imaged on the Ivis Spectrum (Perkin Elmer) using the 745 nm excitation and 820 nm emission filters.
  • the whole tissues were then imaged on the Odyssey CLx near-infrared imaging system (Li-Cor Biosciences) using the 800 nm channel (785 nm excitation).
  • the tumor tissue was frozen on dry ice in Optimal Cutting Tissue medium (OCT) (Tissue Tek), sliced into 12 ⁇ m sections, placed on charged slides (Fisherbrand) and scanned on the Odyssey. The tissue was scanned using the “auto” intensity setting and 21 ⁇ m resolution. Images were analyzed using the Image Studio software (Li-Cor) by measuring the fluorescent signal within a region of interest (ROI) drawn in each tissue image. Slides were stained with Hematoxylin and Eosin (H&E) using standard histological protocols.
  • OCT Optimal Cutting Tissue medium
  • ROI region of interest
  • H&E Hematoxylin and Eosin
  • SNR Signal in tumor compared to muscle
  • tissue thickness In order to standardize tissue thickness, the tissue was sliced in 12 ⁇ m sections, scanned, and analyzed. SNR in the orthotopic samples was 12.7-200 while the SNR for the flank xenograft tissue was 246-344. The higher SNR ratios between whole tissue and sliced tissue analysis is most likely due to the very low levels of signal detected in normal brain tissue which were often below the level of detection in 12 ⁇ m sections.
  • Signal in tumor tissue compared to normal muscle was dose and time dependent.
  • the optimal dose for a one day imaging time point was determined to be 6 nmol while the optimal dose for a three day time point was 20 nmol.
  • Signal in normal tissues distributes rapidly after injection with the highest accumulation of Compound 16 in the kidney, liver, and skin one day after injection. Compound 16 cleared out of normal tissues with residual amounts remaining in the kidney, liver, and skin after three days.
  • Optimal imaging time after Compound 16 injection was assessed for doses between 0.6- and 20 nmol.
  • Mice with U87 flank xenografls were imaged 1, 2 or 3 days after injection.
  • signal in tumor compared to muscle signal to noise ratio, SNR
  • SNR signal to noise ratio
  • Tumor targeting and imaging efficacy of Compound 16 was assessed in the U87 orthotopic brain xenograft mouse model of glioma.
  • Mice with U87 human glioma cells implanted in either the brain or the flank were injected with 10 nmole of Compound 16.
  • Brain and tumor tissue was excised and imaged 1 day after injection.
  • Signal in tumor compared to normal brain was assessed on both whole tissue images and frozen tissue sliced in 12 ⁇ m sections.
  • Signal in tumor compared to normal brain (SNR) was 11.6-60 for the orthotopic xenograft samples and 131-138 for the flank xenograft samples ( FIG. 13 ). Because most of the tumor tissue adhered to the skull, orthotopic sample #1 was not included in the quantitative analysis. Residual tumor cells were detected in the brain ( FIG. 13 ).
  • Compound 16 was detected in tumors from two out of three U87 orthotopic brain tumors using the IVIS Spectrum ( FIG. 15 ).
  • the brain tumor from orthotopic #1 did not have a signal on the Ivis Spectrum.
  • the absence of signal was due to the tumor tissue adhering to the underside of the skull which was subsequently pulled out of the brain during necropsy.
  • Compound 16 signal was detected in the skull during whole tissue imaging on the Odyssey ( FIG. 15 ).
  • This example evaluates the optimal imaging time and dose of Compounds 1-720 in mice and further shows that Compounds 1-720 target U87 human glioma cells implanted into the brains of mice.
  • Compounds 1-720 signal in tumor compared to normal brain (SNR) is compared to the SNR calculated in subcutaneous U87 flank xenografts using both whole tissue imaging and sliced tissue analysis.
  • SNR Signal in tumor compared to muscle
  • Signal in tumor tissue compared to normal muscle was dose and time dependent.
  • the optimal dose for a one day imaging time point was determined to be 6 nmol while the optimal dose for a three day time point was 20 nmol.
  • Signal in normal tissues distributes rapidly after injection with the highest accumulation of Compounds 1-720 in the kidney, liver, and skin one day after injection. Compounds 1-720 clear out of normal tissues with residual amounts remaining in the kidney, liver, and skin after three days.
  • Optimal imaging time after injection of Compounds 1-720 is assessed for doses between 0.6- and 20 nmol.
  • Mice with U87 flank xenografts are imaged 1, 2 or 3 days after injection.
  • signal in tumor compared to muscle signal to noise ratio, SNR
  • SNR signal to noise ratio
  • Tumor targeting and imaging efficacy of Compounds 1-720 is assessed in the U87 orthotopic brain xenograft mouse model of glioma.
  • Mice with U87 human glioma cells implanted in either the brain or the flank are injected with 10 nmole of Compounds 1-720.
  • Brain and tumor tissue is excised and imaged 1 day after injection.
  • Signal in tumor compared to normal brain is assessed on both whole tissue images and frozen tissue sliced in 12 ⁇ m sections.
  • Signal in orthotropic brain tumors is higher than normal brain tissue using whole tissue imaging and sliced tissue analysis.
  • Signal in flank tumors is higher than normal brain tissue using whole tissue imaging and sliced tissue analysis.
  • This example describes the determination of optimal BLZ-100 imaging dose in dogs with naturally occurring tumors. This analysis was conducted using tissues from dogs enrolled in the study described in Example 19. The optimal clinical imaging dose is a function of the overall fluorescence intensity of the tumor, which impacts ease of detection, and the ratio of signal in the tumor tissue compared with the normal tissue in which it resides. Comparison of tumor intensity and signal to background were performed using ex vivo imaging on gross tumors and sections.
  • BLZ-100 was given intravenously at fixed doses of 0.1-1.5 mg in a dose escalation scheme, followed by expansion at the apparent optimal dose. To normalize for variation in body size, doses were computed in mg/m 2 for all dogs. Doses (mg/m 2 ) were 0.25-0.8 (7 dogs), 0.8-1.2 (16) and 1.2-1.6 (5).
  • the Odyssey near-infrared scanner (Li-Cor) is a flat-bed scanner that is optimized for detection of 800 nm fluorescence. It has 21 micron resolution and gives quantitative data for up to 9 orders of magnitude of intensity. This instrument was able to measure fluorescence in even the very early samples from patients treated with the lowest doses. Data from the gross tumor Odyssey scans was used to compare overall fluorescence across all doses administered. The analysis shows that at the low doses, tumor intensity is correlated with dose level. At doses up to 0.8 mg/m 2 , signal in gross tumor samples increased as a function of dose. At doses above 0.8 mg/m 2 , no further gain in fluorescence was apparent under these conditions. Therefore this is the lower limit of the optimal dose range for imaging canine tumors under at least these conditions.
  • Tumor type was the most important variable in imaging intensity at doses greater than 0.8 mg/m 2 .
  • a region of interest analysis was conducted using the Odyssey scans of the gross tumors.
  • a subset of the soft tissue sarcomas had highest overall uptake, followed by the carcinomas (adenocarcinoma and squamous cell carcinoma).
  • Oral fibrosarcomas had the lowest overall uptake, but it was unclear whether this was due to the histologic subtype or to anatomic location.
  • the data show that soft tissue sarcomas, as a class, have the highest fluorescence, with a median intensity almost threefold that of the carcinomas, and a maximum more than 7 fold higher.
  • ratios of fluorescence in tumor to normal surrounding tissue ranged from ⁇ 1 (no specific signal, 3 dogs) to >200, with good differentiation in several tumor types including meningioma, carcinomas (lung, thyroid, and mammary), and sarcomas. Highest signals and gross tumor to background ratios were seen in a subset of soft tissue sarcomas, suggesting preferential uptake of the conjugate in these tumor types.
  • the soft-tissue sarcomas showed the most intense fluorescence on gross imaging, so these tumors were selected to perform a histopathologic analysis of tumor to background ratio for determination of the upper limit of the optimal dose range. It is presumed that when tumor uptake is maximal, further dose administration will result in higher background staining and reduction in tumor to background ratio (TBR). The analysis of soft-tissue sarcomas showed that this was indeed the case.
  • TBR soft tissue sarcoma cases were available for this analysis (Patients 11, 12, 13, and 19). Tissues were sectioned on a cryostat, and 30 micron sections were imaged on the Odyssey scanner. These sections or serial sections were stained with H&E and read by an expert histopathologist who was blinded to the fluorescence data. A grid was overlaid on the fluorescence image, and total fluorescence in each grid square was measured using Image Studio (Li-Cor) software provided with the Odyssey scanner. Overlay of the fluorescence image with the scored H&E image enabled calling of tumor vs. non-tumor for each grid square. The average of the fluorescence intensity across all tumor and non-tumor grid squares in a section was used to compute TBR for each patient. The TBR declined with increasing dose across these four tumor samples, indicating that higher doses may contribute to increased background staining and loss of specificity. The time between dose and imaging may also influence the TBR.
  • Dog 13 (TBR 207) had surgery 48 hours after dose administration, and dogs 11 (TBR 0.44), 12 (TBR 16), and 19 (TBR 4) had surgery 24 hours after dose administration. Excluding dog 13, comparison of the TBR for the 24 hour cases shows the same trend, supporting the overall conclusion.
  • This example describes the determination of optimal chlorotoxin conjugate Compound 16 imaging dose in dogs with naturally occurring tumors.
  • Chlorotoxin conjugate Compound 16 was given intravenously at fixed doses of 0.1-1.5 mg in a dose escalation scheme, followed by expansion at the apparent optimal dose.
  • the total fluorescence within each region of interest (ROI) was plotted as a function of dose ( FIG. 16 ).
  • a subset of the soft tissue sarcomas had highest overall uptake, followed by the carcinomas (adenocarcinoma and squamous cell carcinoma). Oral fibrosarcomas had the lowest overall uptake, but it was unclear whether this was due to the histologic subtype or to anatomic location. Highest signals and gross tumor to background ratios were seen in a subset of soft tissue sarcomas ( FIG. 17 ), suggesting preferential uptake of the conjugate in these tumor types.
  • the soft-tissue sarcomas showed the most intense fluorescence on gross imaging, so these tumors were selected to perform a histopathologic analysis of tumor to background ratio for determination of the upper limit of the optimal dose range.
  • the average of the fluorescence intensity across all tumor and non-tumor grid squares in a section was used to compute TBR for each patient. The results are shown in FIG. 18 .
  • the TBR declined with increasing dose, indicating that higher doses may contribute to increased background staining and loss of specificity.
  • This example describes the determination of optimal imaging dose of Compounds 1-720 in dogs with naturally occurring tumors.
  • Compounds 1-720 are given intravenously at fixed doses, followed by expansion at the apparent optimal dose.
  • Tumor type is the most important variable in imaging intensity at higher doses. There are no associations between signal and other study variables, such as breed and body mass.
  • Soft-tissue sarcomas are selected to perform a histopathologic analysis of tumor to background ratio for determination of the upper limit of the optimal dose range. When tumor uptake is maximal, further dose administration results in higher background staining and reduction in tumor to background ratio (TBR).
  • TBR tumor to background ratio
  • TBR declines with increasing dose for tumor samples, indicating that higher doses may contribute to increased background staining and loss of specificity.
  • the time between dose and imaging may also influence the TBR.
  • This example describes a method for quantitating the fluorescence for various tumor types labeled with BLZ-100. Multiple canine tumor types were explored in this study in order to determine whether specific tumor types are more amenable to imaging with a chlorotoxin conjugate than others, and to gain broad experience with tumors arising in various anatomic locations and tissue types.
  • the soft-tissue sarcomas include a wide variety of histologic types and grades, so the variability seen in this class may be due to variation in histology and/or grade. They also can have high intratumor variability (see below), so some of the variation in the Odyssey data could be due to the sections of tumor that were sampled.
  • Two patients with fibrosarcoma in the group treated with effective imaging doses had tumors arising in the jaw. Since tumors arising in the jaw generally had poor uptake, it is unclear whether the anatomic site played a role in the limited uptake and specificity in these tumors.
  • the intensity values for soft-tissue sarcomas (all subtypes) and the carcinomas (adenocarcinomas and squamous cell carcinomas) were compared for all cases treated with doses at or above 0.8 mg/m 2 . This analysis revealed a high level of variability among soft-tissue sarcomas, and weaker but more consistent intensities among the carcinomas.
  • ratios of fluorescence in tumor to normal surrounding tissue ranged from ⁇ 1 (no specific signal) to >200.
  • This example describes a method for quantitating the fluorescence for various tumor types labeled with chlorotoxin conjugate Compound 16. Multiple canine tumor types were explored. The intensity values for soft-tissue sarcomas (all subtypes) and the carcinomas (adenocarcinomas and squamous cell carcinomas) were compared for all cases treated with doses at or above 0.8 mg/m 2 . This analysis shows the variability among soft-tissue sarcomas, and the lower but relatively consistent intensities among the carcinomas ( FIG. 19 ).
  • Ratios of fluorescence in tumor to normal surrounding tissue ranged from ⁇ 1 (no specific signal) to >200 as shown in Table 34.
  • TBR 5-17 Spindle cell (1): TBR 2 Fibrosarcoma, jaw (2): TBR 0.5-3 (low uptake in tumors, and high background in oral mucosa) Hemangiosarcoma, vertebral body (1): TBR ⁇ 1 Chondrosarcoma (1): non-specific; patient had radiation therapy prior to treatment. Tumor was necrotic and nasal mucosa had high background.
  • This example describes a method for quantitating the fluorescence for various tumor types labeled with Compounds 1-720. Multiple tumor types are explored in this study in order to determine whether specific tumor types are more amenable to imaging with a chlorotoxin conjugate than others, and to gain broad experience with tumors arising in various anatomic locations and tissue types.
  • the soft-tissue sarcomas include a wide variety of histologic types and grades, so the variability seen in this class may be due to variation in histology and/or grade. They also can have high intratumor variability, so some of the variation in the Odyssey data could be due to the sections of tumor that were sampled.
  • the intensity values for soft-tissue sarcomas (all subtypes) and the carcinomas (adenocarcinomas and squamous cell carcinomas) are compared for all cases treated with doses at or above 0.8 mg/m 2 . This analysis shows the variability among soft-tissue sarcomas, and the lower but relatively consistent intensities among the carcinomas.
  • This example shows experimental analysis of tolerability of Compound 16 in normal CD-1 mice.
  • Compound 16 was formulated in 10 mM Histidine, 5% Dextrose at concentrations of 5, 0.5, 0.05 mg/ml.
  • Chlorotoxin free peptide (KNT-01) was manufactured by Alamone Laboratory and formulated in 10 mM Histidine, 5% Dextrose at a concentration of 1 mM (200 nmole/200 ⁇ l).
  • Compound 76 free peptide (KNT-02) was manufactured by American Peptide Company and formulated in 10 mM Histidine, 5% Dextrose at a concentration of 1 mM (200 nmole/200 ⁇ l) (the peptide component of Compound 76 having a sequence of H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Ala-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Ala-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH).
  • Compound 16 free peptide was manufactured by Bachem and formulated in 10 mM Histidine, 5% Dextrose at concentrations of 1 mM, 0.1 mM and 0.01 mM (the peptide component of Compound 16 having a sequence of H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Arg-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH).
  • mice 6-10 week old female CD-1 mice were injected with 2, 20, or 200 nmol of Compound 16 diluted in 200 ⁇ l of 10 mM Histidine, 5% Dextrose in the tail vein. These dose levels are equivalent to 0.01, 0.1 and 1 mg of conjugate, respectively. Vehicle only was used as the control group. Mice were observed 10 min, 1 hour, and 4 hours after injection and then daily until euthanasia at 3 or 14 days post dose. Mice were scored using a modified Body Condition Score (BCS) and activity level by visual inspection. Body weight was measured every 3 days. Blood was collected using terminal cardiac puncture and placed in serum separating tubes (SST microtainer). General chemistry screens were performed by Phoenix Central Laboratory.
  • BCS Body Condition Score
  • mice 6-10 week female CD-1 mice were injected with 0.008, 0.08, or 0.8 mg (molar equivalent of conjugate) of native chlorotoxin (KNT-01), Compound 76 (KNT-02), or Compound 16 (KNT-03) free peptide diluted in 200 ⁇ l of 10 mM Histidine, 5% Dextrose in the tail vein. The mice were observed for 1 hour post injection for activity level.
  • KNT-01 native chlorotoxin
  • KNT-02 Compound 76
  • KNT-03 Compound 16 free peptide diluted in 200 ⁇ l of 10 mM Histidine, 5% Dextrose in the tail vein.
  • the mice were observed for 1 hour post injection for activity level.
  • mice were evaluated 10 minutes, 1 hour, and 4 hours after injection then once daily until euthanasia 3 or 14 days post treatment. Mice in the vehicle control and the 0.01 mg dose group were normal at all observation time points.
  • Four out of six mice in the 0.1 mg group and six out of six animals in the 1 mg dose cohort exhibited a decrease in spontaneous motor activity, somnolence, and prostration approximately 1-3 minutes after the injection. Ptosis was noted in some mice. The hypoactive behavior lasted 30-60 minutes and all mice had completely recovered by the 4 hour observation time point. The mice were not unconscious or paralyzed. Breathing remained normal. Coloration remained normal without signs of cyanosis, red eyes, or lacrimation. The decrease in motor activity behavior lasted approximately 30-60 minutes after injection.
  • mice were injected with the KNT-03 free peptide, or the related KNT-01 or KNT-02 free peptides. Similar to the Compound 16 conjugate, all of the mice injected with free peptides in the high dose (200 nmol) showed a decrease in activity level, somnolence, and prostration starting 3 minutes post injection. This indicated that the hypoactive effect resulted from the peptide backbone. Because the effect was observed in the free peptide with the native sequence, the transient hypoactivity was not a novel property created by the mutated Compound 16 peptide and/or conjugation to the dye. In addition, this transient behavior was only observed in mice. Rats and non-human primates injected with similar high doses of Compound 16 did not exhibit abnormal activity levels.
  • mice were clinically normal by visual inspection for the duration of the study. All mice had BCS3 scores at each health observation indicating the mice were healthy. Mice were weighed every three days until euthanasia. No dose related changes in body weight were observed ( FIG. 67 ).
  • This example shows experimental analysis of tolerability of Compounds 1-720 in normal CD-1 mice.
  • mice are evaluated 10 minutes, 1 hour, and 4 hours after injection then once daily until euthanasia 3 or 14 days post treatment. Some mice exhibit a decrease in spontaneous motor activity, somnolence, and prostration approximately 1-3 minutes after the injection. In order to ascertain if the decrease in motor activity level is due to Compounds 1-720, additional mice are injected with the free peptides. All of the mice injected with free peptides show a decrease in activity level, somnolence, and prostration starting 3 minutes post injection. This indicates that the hypoactive effect results from the peptide backbone. Because the effect is observed in the free peptide with the native sequence, the transient hypoactivity is not a novel property created by Compounds 1-720 and/or conjugation to the dye. In addition, this transient behavior is only observed in mice. Rats and non-human primates injected with similar high doses of Compounds 1-720 do not exhibit abnormal activity levels.
  • mice are clinically normal by visual inspection for the duration of the study. All mice have BCS3 scores at each health observation indicating the mice are healthy. Mice are weighed every three days until euthanasia. No dose related changes in body weight are observed.
  • This example shows targeting and illumination of medulloblastoma tumors and cells in a ND2:SmoA1 mouse model using Compound 16 and further shows clinical and pathological effects of high doses of Compound 16.
  • Medulloblastoma is the most common malignant solid tumor in children.
  • Current therapy includes maximal safe surgical resection, irradiation, and chemotherapy.
  • Complete surgical resection of the tumor heavily influences the prognosis of patients with medulloblastoma by conferring a 30% survival improvement over patients with residual disease.
  • Patients with residual disease are considered high-risk for tumor progression and are treated with higher doses of radiation and chemotherapy. These patients have a greater risk of suffering from the deleterious side-effects of aggressive treatments while facing a lower chance of surviving their disease.
  • the goal of near-complete surgical resection must be balanced with surgically accurate tumor removal because damaging healthy brain tissue could severely impair normal neurological functions.
  • Strategies, such as Compound 16 guided surgery are needed to improve complete and accurate tumor resection in patients with brain cancer increasing patient survival and reducing morbidity.
  • the ND2:SmoA1 (abbreviated SmoA1) mouse model of medulloblastoma on a C57BL/6 background was used to evaluate binding of Compound 16 to medulloblastoma tumor. These mice develop spontaneous medulloblastoma tumors in the cerebellum that closely resemble the human disease.
  • These genetically engineered transgenic mice express a constitutively active smoothened mutant protein (SmoA1) driven by a 1-kb fragment of the neuroD2 promoter. This promoter is activated mainly in the cerebellar granule neuron precursors in the brain.
  • This mouse model mimics the sonic-hedgehog pathway subtype of medulloblastoma.
  • Symptomatic mice that were homozygous for the transgene were selected for enrollment in these studies. Clinical symptoms of brain cancer were detected using an open field cage evaluation. Symptoms include head tilt, hunched posture, ataxia, protruding skull, and weight loss.
  • IVIS Spectrum Perkin Elmer
  • ROI's regions of interest
  • mice with moderate symptoms of medulloblastoma received an intravenous injection of 10 nmol of Compound 16.
  • Mice were administered 100 ⁇ l of 0.5 mg/ml (10 nmol/100 ⁇ l) Compound 16 through the tail vein.
  • One day after injection the mice were euthanized using CO2 inhalation.
  • Ex vivo whole brain imaging was performed with the IVIS Spectrum (Perkin Elmer) using the 745 nm/820 nm excitation and emission filter set, and on the Odyssey CLx (Li-Cor Biosciences) near-infrared scanner using the 800 nm setting (785 nm excitation laser).
  • the tissue was then frozen in Optimal Cutting Temperature medium (OCT, Tissue Tek) on dry ice or fixed in 10% neutral buffered formalin.
  • OCT Optimal Cutting Temperature medium
  • the frozen tissue was sliced into 12 ⁇ m sections.
  • Tissues were either scanned using the Odyssey CLx scanner or stained with hematoxylin and eosin (H&E) according to standard histological protocols.
  • H&E hematoxylin and eosin
  • the tissue was processed, embedded, sliced, and H&E stained according to standard protocols at Histology Consultation Services. Slides were scanned on the Aperio Scanscope AT (Leica Biosystems) at 20 ⁇ magnification.
  • ROI region of interest
  • SNR normal tissue
  • SmoA1 mice with medulloblastoma were injected with Compound 16 and imaged one day later.
  • Compound 16 was higher in tumor tissue compared to normal brain in all eight of the SmoA1 mice that were enrolled in the study.
  • Signal in tumor tissue compared to normal brain was between 5.9 and 47.
  • Four out of eight samples had considerably lower signal in the tumor which was near the lower level of detection for the IVIS Spectrum and the Odyssey scanner.
  • Signal in unaffected cortex was 2.7-3.6 fold higher and signal in normal brains from Nu/Nu mice was the same as non-injected animals. Small foci of metastatic leptomeningeal spread were detected using Compound 16.
  • the higher signal was possibly caused by the higher dose of Compound 16 that was used in SmoA1 mice or abnormalities that affect the entire SmoA1 brain.
  • the SmoA1 mice often have mild to severe hydrocephalus which often affects clearance of Compound 16 from non-tumor tissue.
  • Leptomeningeal spread in SmoA1 mice was occasionally observed. These mice, like 30-35% of human patients, develop small foci of tumor cells in the meningeal membrane. Leptomeningeal spread was detected in one mouse using Compound 16.
  • a whole brain fluorescent scan of SmoA1-1 is shown ( FIG. 21 ). The scan illuminates small foci of fluorescence that corresponds to small clusters of tumor cells highlighted in the H&E stained slide ( FIG. 21 ).
  • mice with medulloblastoma had a higher signal in the tumor than normal cortex with SNR's ranging from 5.9-47 after administration of Compound 16. While the signal in tumor was higher than normal in all mice, four of the samples had noticeably lower signal in the tissue. Compound 16 signal was not detected in mice with normal brain tissue (Nu/Nu mice) while signal in unaffected SmoA1 cortex was 2.7-3.6 fold higher than the non-injected animals. The residual background signal in non-tumor tissue is possibly caused by hydrocephalus which occurs often in these mice. Small foci of metastatic cells were detected using Compound 16 in one mouse emphasizing Compound 16's capability to detect even small clusters of cancerous tissue.
  • This example shows targeting and illumination of medulloblastoma tumors and cells in a ND2:SmoA1 mouse model using Compounds 1-720 and further shows clinical and pathological effects of high doses of Compounds 1-720.
  • the ND2:SmoA1 (abbreviated SmoA1) mouse model of medulloblastoma on a C57BL/6 background is used to evaluate binding of Compounds 1-720 to medulloblastoma tumor.
  • SmoA1 mice with medulloblastoma are injected with Compounds 1-720 and imaged one day later.
  • Compounds 1-720 is higher in tumor tissue compared to normal brain in all eight of the SmoA1 mice that are enrolled in the study. Background signal in normal brain due to non-specific binding or incomplete clearance is evaluated in mice that have been injected with 6 nmol of Compounds 1-720.
  • Compounds 1-720 is sufficiently cleared from normal brain tissue without non-specific binding one day after injection.
  • Compounds 1-720 analyzed in tumor bearing SmoA1 mice has a higher signal in the tumor than the normal cortex.
  • mice with medulloblastoma have a higher signal in the tumor than normal cortex.
  • Compounds 1-720 signal is not detected in mice with normal brain tissue (Nu/Nu mice) while signal
  • Compounds 1-720 emphasizing the capability of Compounds 1-720 to detect even small clusters of cancerous tissue.
  • This example describes methods for detecting naturally occurring solid tumors in dogs using the fluorescently labeled chlorotoxin conjugate, BLZ-100.
  • canine tumors resemble human disease, including sarcomas, breast and lung cancers, mucosal squamous cell cancers, and gliomas.
  • the diversity of these spontaneously occurring tumors in size and type, surrounding tissue, and patient body mass provides a model that is superior to the mouse in predicting the clinical characteristics of a chlorotoxin conjugate, such as BLZ-100, including tumor penetration, background staining, and effective imaging dose.
  • Dogs received standard of care including tumor resection with intent to control or cure local disease. Dogs received BLZ-100 intravenously 24-48 hours before surgery, and tissues were imaged ex make after surgery. Ex vivo imaging was performed on gross tissue specimens using the IVIS Spectrum (PerkinElmer) and the Odyssey NIR scanner (Li-Cor) to determine overall signal in tumor and gross signal to background. Tissues were embedded in OCT, sectioned on a cryostat, and scanned on the Odyssey. Serial sections were stained with H&E, and comparison with the fluorescence scans was used to validate the specificity of BLZ-100 for tumor tissue. Intraoperative imaging was conducted in several cases, using a prototype open NIR imaging device.
  • BLZ-100 was prepared in a formulation buffer (100 mM histidine/5% dextrose, 10 mM Tris/5% dextrose, or 10 mM Tris/5% mannitol).
  • a formulation buffer 100 mM histidine/5% dextrose, 10 mM Tris/5% dextrose, or 10 mM Tris/5% mannitol.
  • lyophilized conjugate was suspended in formulation buffer.
  • the material was drawn up into a sterile syringe, and then aliquoted through a sterile 0.44 micron filter into pre-capped sterile amber glass vials. The vials were stored at ⁇ 20° C., and were shipped on dry ice to the study site.
  • Tumor types included several subtypes of sarcoma; oral and cutaneous squamous cell carcinomas; mast cell tumors; adenocarcinomas including lung, mammary, and thyroid; and a brain meningioma.
  • BLZ-100 was given intravenously 24-48 hours prior to surgery. Complete blood count, serum chemistry, and urinalysis data from prior to, and 24-48 hours post injection, were evaluated for changes that might indicate toxicity from the CTX. There were small declines in serum BUN, calcium, and potassium levels and in urine pH that reached statistical significance (two-tailed t-test); however, they remained well within the normal ranges and were not considered clinically significant. There were no other significant differences, and no overt safety concerns noted.
  • Serum samples were collected at time points following dose administration. A fluorescence assay was used to calculate the serum concentration of BLZ-100. The data show a rapid distribution into tissues, followed by a slower elimination phase. These data are similar to those obtained from laboratory mice, rats, dogs, and non-human primates.
  • This example describes methods for detecting naturally occurring solid tumors in dogs using Compound 16.
  • Serum samples were collected at time points following dose administration. A fluorescence assay was used to calculate the serum concentration of Compound 16. The data show a rapid distribution into tissues, followed by a slower elimination phase (Table 37).
  • Serum Compound 16 levels in dogs measured by standard curve analysis of fluorescence at time points following dosing. Serum samples from each time point were diluted 1:1 in formulation buffer. A standard curve of Compound 16 (10 mcg/ml to 4 ng/ml) in 50% serum/50% formulation buffer was prepared. Fluorescence was measured on the Odyssey scanner (765 nm excitation/800 nm emission). Serum concentrations of the product were back-calculated using the standard curve.
  • Patient Dose ( mg/m 2 ) Calculated serum BLZ-100 concentration, ng/ml 1 0.44 520.49 31.51 2.21 1.30 2 0.28 91.78 332.41 28.63 1.53 3 0.25 1358.79 105.62 61.22 11.87 4 0.30 140.20 48.15 11.42 2.91 5 0.50 140.70 42.22 14.96 2.31 6 0.56 161.12 42.53 6.93 1.20 7 0.83 262.08 56.84 14.04 1.37 9 1.03 2277.50 103.09 84.62 1.14 10 1.12 634.11 206.93 12.78 2.92 11 1.62 1685.95 620.00 1.31 12 1.06 8243.41 716.46 17.08 4.92 13 0.94 862.19 149.60 14.84 0.69 14 1.06 1561.45 193.46 20.00 0.53 15 1.21 1668.98 121.85 20.70 0.95 16 1.26 405.91 93.
  • This example describes methods for detecting naturally occurring solid tumors in dogs using Compounds 1-720.
  • Serum samples are collected at time points following dose administration.
  • a fluorescence assay is used to calculate the serum concentration of Compounds 1-720.
  • the data show a rapid distribution into tissues, followed by a slower elimination phase. These data are similar for laboratory mice, rats, dogs, and non-human primates.
  • PK pharmacokinetic
  • IV intravenous
  • PK samples from these studies were analyzed using research-based methods.
  • the PK of Compound 16 following IV administration was evaluated as part of GLP single dose toxicology studies in rats and monkeys.
  • serum Compound 16 concentrations were determined using validated LC/MS-based methods.
  • Compound 16 was intravenously administered as a single-use intraoperative fluorescent imaging agent to specifically label tumor tissue.
  • Compound 16 was sterilely formulated in a liquid at 2 mg/mL in Tris/mannitol buffer at neutral pH. The dye was chemically linked via a single lysine residue on the CTX peptide.
  • Compound 16 contained no novel excipients or linker molecules.
  • a tabular listing of the single-dose nonclinical toxicity studies conducted to support initiation of first-in-human (FIH) clinical trials of Compound 16 is provided in Table 38.
  • All species used for nonclinical safety evaluations were pharmacologically relevant for Compound 16 based on a high degree of sequence homology to the CTX peptide target.
  • the rat and monkey were selected as the primary species for GLP toxicology studies based on the sequence homology to the CTX peptide target (97 and 100% respectively), suitability of the species for safety assessment (i.e., preference for rat over mouse), and lack of potential confounding effects (pseudoallergy/hypersensitivity reactions have been observed in the dog but not in the monkey).
  • Toxicology studies were performed in accordance with Good Laboratory Practice (GLP) regulations.
  • GLP Good Laboratory Practice
  • ICH International Conference on Harmonization
  • FDA Food and Drug Administration
  • dose levels and method of administration were based on initial mouse pharmacology models in which a dose level of 0.01 mg produced consistent tumor imaging. Since the preferred method of dose administration in the clinical trials is on a fixed basis (i.e., not adjusted for body weight or surface area), dose levels in the nonclinical safety studies were converted to fixed dose levels using estimated body surface area for each species. The dose level for humans was estimated using imaging data in mice and dogs (Table 39).
  • mice displayed transiently decreased spontaneous motor activity, somnolence and prostration following intraveneous (IV) administration of 0.1 and 1 mg of Compound 16.
  • Transient salivation was the only finding in the pilot study in male rats following IV administration of 0.03, 0.3, or 1.5 mg Compound 16 0 IV.
  • notable findings in the pilot study included pseudoallergy/hypersensitivity reactions (i.e., itching/scratching, warm ears, etc.) in 2 of 2 treated dogs during or immediately after IV administration of 1 mg Compound 16. The mechanism of the pseudoallergy has been further explored in 3 male beagle dogs following IV administration of 1 mg of Compound 16.
  • mice were clinically normal during all observation time points. A subset of mice at 0.1 mg and all mice at 1 mg exhibited a decrease in spontaneous motor activity, somnolence, and prostration occurring as rapidly as 1 minute post-dose. The hypoactivity lasted approximately 30 to 60 minutes. All mice returned to normal by the 4 hour observation time point and remained normal for the duration of the study. Similar clinical signs were seen when mice were injected with equimolar amounts of the peptide backbone of Compound 16.
  • Transient treatment-related clinical signs were limited to salivation, which was observed at the 0.03 mg dose in 2 rats at 20 minutes and 1 hour post-dose, at the 0.3 mg dose in 2 rats at 20 minutes post-dose, and at the 1.5 mg dose in 1 rat at 20 minutes, 1 hour, and 2 hours post-dose.
  • the gross pathology findings were limited to green discoloration of the kidneys on days 3 and 15 of the study. As noted above, there were no Compound 16-related findings on organ weights or microscopic findings at any dose level. The findings of green discoloration in kidneys are not considered adverse. As such, the NOAEL was considered the high dose of 7 mg, or approximately 28 mg/kg.
  • Compound 16 is a biotechnology-derived pharmaceutical candidate that is formulated with commonly-used excipients, such as Tris buffer and D-mannitol.
  • excipients such as Tris buffer and D-mannitol.
  • no treatment-related hematological findings were identified in the completed nonclinical safety studies or in the preliminary data from the on-going phase 1 clinical safety study in subjects with skin cancer and (2) no irritation or lesions at the injection site were identified by macroscopic examination or by histopathology in the single-dose, intravenous administration, or toxicology studies in rats and monkeys.
  • the peptide component of Compound 16 is similar to native CTX.
  • a synthetic version of the CTX peptide (TM-601) has been studied in mice and marmosets and it was well-tolerated.
  • the NOAEL for TM-601 after a single IV dose in the mouse was 6.4 mg/kg (highest dose tested) and 2.0 mg/kg in the marmoset (highest dose tested).
  • Repeated-dosing for 7 weeks in mice at doses of 2 and 5 mg/kg by intravenous administration resulted in clinical signs of transient ptosis and hypoactivity within 1 hour post-dose. No effects on hematology or tissue pathology were observed.
  • ICG insulin receptor agonist
  • Compound 16 contains roughly 0.15 mg of dye per mg of drug product.
  • Compound 16 imaging doses are currently estimated to range from 3 to 12 mg, or 0.45 to 1.8 mg equivalents of ICG.
  • LC/MS methods were developed and used to analyze serum from a non-GLP rat study and from GLP studies in rats and cynomolgus monkeys. The LC/MS methods were validated prior to GLP study sample analyses.
  • the Lowest Level of Quantitation (LLOQ) for Compound 16 was 10 ng/mL and 5 ng/mL in rat and monkey serum, respectively. The studies in which these methods were used are listed in Table 43.
  • the stability of rat serum samples stored at ⁇ 70° C. was at least 9 months. Stability data from the monkey support serum sample storage up to 1 month at ⁇ 70° C. Incurred sample reanalysis (ISR) was performed on samples from the rat and monkey GLP studies. The rat ISR assessment passed, however the monkey ISR did not.
  • ISR Incurred sample reanalysis
  • follow-up investigations of the ISR failure in the monkey study were conducted in an attempt to identify a root cause and assess impact of the ISR failure on the study data. No single root cause was identified, however issues with sample stability and variability from the various lots of control serum and matrix likely contributed. It was noted that the failure rate was highest in samples with relatively low concentrations of Compound 16.
  • the internal standard used in the human method is an isotope-labeled version of Compound 16 which is approximately 30 Da heavier than the preclinical standard, Compound 76 (the peptide component of Compound 76 having a sequence of H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Ala-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Ala-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH).
  • bovine serum albumin has been added to the standards and quality control solutions to improve stability.
  • an AB Sciex QTrap 5500 mass spectrometer was used in place of the Sciex API 5000.
  • the LLOQ for the human assay is 10 ng/mL.
  • Compound 16 serum concentration versus time data were downloaded into Phoenix WinNonlin 6.3 (Pharsight, Cary, N.C.) for analyses using standard noncompartmental methods of intravenous bolus, intravenous infusion, or extravascular input as appropriate.
  • Mouse and rat PK data were analyzed using the mean serum concentration versus time data.
  • Dog and monkey PK data were analyzed by individual animal and then group summary statistics were calculated. Samples that were not analyzed for Compound 16 concentration were due to insufficient sample volume or concentration values below the limit of quantitation of the assay. Such samples were treated as missing for the purpose of calculating toxicokinetic (TK) parameters, and were not included in the calculation of group means. Nominal dose and sample collection times were used in estimating parameters.
  • TK toxicokinetic
  • Compound 16 was administered IV as the intended clinical route of administration is IV.
  • mice Female mice were intravenously or subcutaneously administered a single fixed dose of 0.02 mg of Compound 16.
  • Compound 16 was formulated in 10 mM Tris/5% dextrose. Serum was collected from mice at 0.25, 2, 6 and 24 hours following administration ( FIG. 22 ). A fluorescence-based method was used to measure Compound 16 serum concentrations.
  • TK Toxicokinetics
  • Biodistribution was evaluated by measuring fluorescence intensity in tissue sections from the organs collected at euthanasia 48 hours after intravenous administration. Kidney, liver, heart, and aorta were collected and fixed in 10% formalin. Tissue was stored at 4° C. in 10% formalin until processed. The tissue was washed twice in PBS then processed through a sucrose gradient of 10% sucrose/PBS for 5 hours followed by 20% sucrose/PBS overnight at 4° C. for cryoprotection. The tissue was then gross sectioned and frozen in OCT on dry ice. The frozen tissue blocks were sliced into 12 ⁇ m sections and placed on gelatin coated slides.
  • the signal emitted from Compound 16 was highest in the kidney which was consistent with renal clearance of the product. Less intense fluorescence was observed in liver and aorta. Signal was low in the heart. Signal increased with dose escalation in all of the tissues that were tested; however, the signal in the heart remained low even at high doses. Signal in the aorta and the great vessels of the heart was relatively high compared to the heart and increased with dose escalation. Distribution of Compound 16 to the kidney, liver, heart, and aorta did not seem to have toxicological significance. There were no corresponding changes in serum chemistry or histopathology.
  • Two male beagle dogs were intravenously administered a nominal dose of 1 mg of Compound 16.
  • the first dog received the dose as an IV bolus and the second dog received the dose as a 15 minute IV infusion.
  • Compound 16 was formulated in 10 mM Tris, 5% Mannitol, pH 7.2. Animals were bled for PK analysis at pre-dose, 0.083, 0.25, 0.5, 1, 2, 4, 6, 24, 48, 72, and 96 hour post-dose (bolus) or post-start of infusion.
  • a fluorescence-based method was used to measure Compound 16 serum concentrations. (Table 44).
  • Compound 16 was administered a nominal dose of 0.6 mg of Compound 16 as an IV bolus.
  • Compound 16 was formulated in 10 mM Tris, 5% Mannitol, pH 7.2. Animals were bled for PK analysis at predose, 0.083, 0.25, 0.5, 1, 2, 4, 6, 24, 48, 72, and 96 hours postdose. A fluorescence-based method was used to measure Compound 16 serum concentrations ( FIG. 24 ).
  • the shape of monkey NHP1's serum concentration versus time profile was not what would be expected following a single IV bolus dose (Table 44). The reason is unknown and these data could not be used to calculate PK parameters that require definition of the terminal phase of the concentration versus time profile.
  • the t 1/2 for NHP2 was approximately 55 hours, CL was 170 mL/hr, and V ss was 6580 mL.
  • Compound 16 Male and female Sprague Dawley rats were administered a single IV bolus dose of Compound 16 at fixed doses of 0.07, 0.7, and 7 mg.
  • Compound 16 was formulated in 10 mM Tris, 5% Mannitol, pH 7.2.
  • PK samples were obtained using a staggered sampling scheme of 3 males and 3 females per timepoint per group at pre-dose, 0.25, 1, 3, 6, 12, 24, and 48 hours post-dose ( FIG. 25 ). Serum samples were analyzed for Compound 16 concentration via a validated LC/MS based procedure and the resulting concentration versus time data were used to estimate TK parameters using non-compartmental analysis.
  • Compound 16 was formulated in 10 mM Tris, 5% Mannitol, pH 7.2.
  • PK samples were obtained pre-dose, 0.083, 0.25, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, and 120 hours post-dose. Serum samples were analyzed for Compound 16 concentration using a validated LC/MS based procedure and the resulting concentration versus time data were used to estimate TK parameters using non-compartmental analysis.
  • Exposure based on C max and C 0 generally increased in a dose-proportional manner, although the 6 mg dose level had higher than expected C max and C 0 values. Exposure based on AUC 0-t values increased in a greater than dose-proportional manner across all dose groups, suggesting that Compound 16 clearance is reduced at higher doses (see Table 46). This also might be due, in part, to an incomplete characterization of the TK profile at the lowest dose level due to assay limitations. There also appeared to be a slower rate of decline in Compound 16 concentrations between 0.25 and 4 hours post-dose in the 6 mg dose group relative to the 0.6 mg dose group.
  • TK parameters were able to be estimated for the 60 mg dose group. There were no substantial differences in the TK parameters between males and females.
  • the overall mean t 1/2 , CL, and V ss were 33.7 hour, 50.6 mL/hr, and 211 mL, respectively.
  • Urine excretion has not been formally assessed. Based on biodistribution data in select normal tissues from single dose mouse tumor models and a non-GLP single dose rat study, the kidney appears to be an important organ involved in the clearance and elimination of Compound 16. This is further supported by data from the single-dose GLP rat study in which green discolored kidneys were noted in the highest dose group 2 days after injection, and the appearance of green colored urine in the single-dose GLP monkey study.
  • Compound 16 PK profiles following IV administration demonstrated a bi-exponential decline with a rapid initial phase and a longer terminal phase in all species.
  • Compound 16 exposure based on C 0 or C max values appeared to be approximately dose proportional over the tested dose ranges.
  • AUC values increased in a dose-proportional or higher than dose-proportional manner, suggesting that Compound 16 clearance is reduced at higher doses.
  • the terminal phase could be characterized in the high dose group (60 mg) in cynomolgus monkeys, allowing estimation of t 1/2 , CL, and V ss parameters.
  • the mean t 1/2 , CL, and V ss values were 33.7 hours, 50.6 mL/hr, and 211 mL, respectively.
  • PK pharmacokinetic
  • Cynomolgus monkeys are intravenously administered a single bolus injection of Compounds 1-720 at a fixed dose level. Clinical observations are made at each time point.
  • PK pharmocokinetic
  • mice, rats, dogs and monkeys are intravenously administered as an IV bolus or IV infusion of Compounds 1-720. Serum is collected at multiple time points following administration. A fluorescence-based method is used to measure Compounds 1-720 serum concentrations. C max , C 0 , t 1/2 and AUC 0-t values are obtained from the resulting data. Biodistribution is evaluated by measuring fluorescence intensity in tissue sections from the organs collected at euthanasia at time-points following intravenous administration. Distribution of Compounds 1-720 to the organs typically does not seem to have toxicological significance, with no corresponding changes in serum chemistry or histopathology.
  • This example demonstrates the pharmacokinetics of Compound 16 in human subjects with nonmelanotic skin cancer.
  • the primary objective of the study was to evaluate the safety and tolerability of a single IV administration of Compound 16.
  • This example demonstrates the pharmacokinetics of a chlorotoxin conjugate in human subjects.
  • IV intravenous
  • bolus injections 1 mg, 3 mg, 12 mg or 30 mg of a chlorotoxin conjugate such as Compound 16.
  • Samples are analyzed with fluoresence-based methods and with liquid chromatography/mass spectrometry (LC/MS) method to determine pharmacokinetic profiles of chlorotoxin conjugate in humans ( FIG. 27 ).
  • LC/MS liquid chromatography/mass spectrometry
  • This example describes the tumor-binding specificity of BLZ-100 and the ratio between tumor and background binding by BLZ-100.
  • a prototype intraoperative imaging system was utilized for patients 17 through 28 from the study described in Example 9. Its use during surgeries enabled imaging of tumor beds as well as tumors in situ and immediately following excision. The data show generally good discrimination between gross tumor and surrounding tissues. Peritumoral skin tended to have background fluorescence, while uninvolved skin had lower fluorescence. Mucosal tissues also showed background fluorescence, resulting in lack of specificity and residual non-tumor fluorescence in patient 17. Tumor bed imaging showed little or no background staining in “internal” tissues such as trachea, muscle, and fat. The intraoperative imaging showed good subjective concordance with the quantitative ex vivo image analysis conducted using the Odyssey scanner. The tumors that had overall high intensity and good tumor to normal ratios ex vivo also showed high contrast and were easy to detect intraoperatively (Table 49).
  • Patient 19 had a grade II soft-tissue sarcoma on her foreleg.
  • the tumor had been clinically evident for several months without treatment.
  • the peritumoral skin was swollen and ulcerated ( FIG. 28A ).
  • Intraoperative imaging of the tumor in situ showed variable fluorescence in the tumor, some fluorescence in the swollen and ulcerated peritumoral skin, and little or no background fluorescence in other areas ( FIGS. 28B, 28C ). Imaging of the tumor immediately post excision showed roughly 8-fold variability of fluorescence intensity within the tumor ( FIGS. 28D, 28E ). The variation within the tumor is consistent with the pathology report that approximately 50% of the mass is replaced by eosinophilic debris (necrosis).
  • Imaging of the tumor bed showed fluorescence in peritumoral skin; a sample of this skin was resected and sent for further imaging and histopathology. There was no residual fluorescence in the tumor bed or in the surrounding uninvolved skin ( FIG. 28F ). A sample of resected peritumoral skin was sent for further imaging and histopathology, which confirmed the absence of neoplastic invasion.
  • Patient 22 had a recurrent mammary adenocarcinoma.
  • the tumor was removed en bloc with overlying skin and surrounding fatty tissue. Imaging of the tumor from the bottom showed that the mass is detectable through ⁇ 0.5 cm of normal fatty tissue ( FIG. 29A ).
  • the diffuse appearance of the fluorescence is due to tissue scattering of the emitted light.
  • a slice for further imaging was removed from the skin side, leaving the bulk of the mass and surrounding tissue exposed.
  • the contrast between the tumor and surrounding tissue is improved due to the absence of intervening tissue ( FIGS. 29B, 29C ).
  • the tissue pieces collected for further imaging contained gross tumor (white areas, FIG. 29D ) and adjacent tissue. Fluorescence imaging shows about 2.5-fold brighter fluorescence in the gross tumor areas compared with the adjacent tissue ( FIG. 29E ). The tumor bed showed no residual fluorescence ( FIG. 29F ). Note that the fluorescence in the skin appears brighter in panel F than in panel C; this is due to the increased sensitivity used in the survey of the tumor bed, to ensure that any residual fluorescence would be detected.
  • Patient 23 had a cutaneous squamous cell carcinoma of the tail.
  • the lesion had penetrated the skin, which was grossly swollen and ulcerated ( FIG. 30A ).
  • the lesion was covered by a serocellular crust.
  • Preoperative fluorescence imaging showed very little fluorescence penetrating the serocellular crust, while the peritumoral skin showed relatively bright staining ( FIG. 30B ).
  • Two “fingers” of fluorescence were noted, which extended to the opposite side of the tail ( FIG. 30C ).
  • FIGS. 30D, 30E at right
  • the remaining central tumor viewed from the side rather than through the serocellular crust, showed fluorescence intensity similar to that of the central tumor.
  • the peritumoral skin was less intense than the tumor itself ( FIG. 30F ), but was about 3-fold more intense than uninvolved skin. Samples of skin from the fluorescent areas on the opposite side of the tumor were submitted for histopathology, and they did not contain tumor.
  • Patient 20 had a thyroid carcinoma.
  • the entire thyroid gland was removed, along with an enlarged lymph node. Intraoperative imaging of the thyroid showed most of the gland was fluorescent, with about 2-fold variation in signal intensity throughout ( FIG. 31 ).
  • the lymph node had regions of fluorescence that were comparable to the primary tumor.
  • the tumor bed had no residual fluorescence. Additionally, no significant non-specific background fluorescence was observed in the internal structures including the trachea, nerves, and arteries. Histopathology showed that the thyroid was 95% effaced by the tumor, which had large areas of blood filled spaces and necrosis. These may account for the variability in staining seen in the primary tumor.
  • the lymph node was confirmed to contain metastatic disease.
  • This example describes the tumor-binding specificity of Compounds 1-720 and the ratio between tumor and background binding by Compounds 1-720.
  • Example 26 Methods and materials used are as described in Example 26, but with Compounds 1-720.
  • a prototype intraoperative imaging system is utilized. Its use during surgeries enables imaging of tumor beds as well as tumors in situ and immediately following excision. The data show generally good discrimination between gross tumor and surrounding tissues. Peritumoral skin tends to have background fluorescence, while uninvolved skin has lower fluorescence. Tumor bed imaging shows little or no background staining in “internal” tissues such as trachea, muscle, and fat. The intraoperative imaging shows good subjective concordance with the quantitative ex vivo image analysis conducted using the Odyssey scanner. The tumors that have overall high intensity and good tumor to normal ratios ex vivo also show high contrast and are easy to detect intraoperatively.
  • This example describes the evaluation of tumor and adjacent tissues at the cellular level in order to assess sensitivity and specificity of a chlorotoxin conjugate, such as BLZ-100 for cancer cells.
  • the data were grouped by individual section and plotted for each patient.
  • the subcutaneous soft tissue sarcomas showed highly specific tumor fluorescence.
  • a logistic regression analysis was used to determine a reasonable threshold intensity for detecting tumor in the subcutaneous soft-tissue sarcomas.
  • Non-skin tissues were used to compute sensitivity and specificity, with a threshold intensity of 30,000 used as a cutoff value.
  • Grid squares were called tumor or no tumor based on fluorescence intensity and based on pathologist call. Concordant and discordant calls are used to calculate sensitivity and specificity. Sensitivity (95%) and specificity (85%) were very good using a threshold grid square fluorescence of 30,000.
  • the cutaneous squamous cell carcinomas had fluorescence signal coming both from the tumor and from the underlying dermis. In most cases, the signal was brighter in the underlying dermis, leading to the “inverted” tumor vs. normal intensity. Although histologic specificity in these tumors is low, truly uninvolved skin seen during intraoperative imaging in patient 23 and during ex vivo imaging in patient 14 was not fluorescent. Analysis of the mammary tumors shows that, like skin, mammary tissue adjacent to tumor takes up BLZ-100. In both tumor types, gross tumors had higher fluorescence than uninvolved tissue.
  • Tissues were sectioned on a cryostat, and 30 micron sections were imaged on the Odyssey scanner. These sections or serial sections were stained with H&E and read by an expert histopathologist who was blinded to the fluorescence data.
  • a grid was overlaid on the fluorescence image, and total fluorescence in each grid square was measured using Image Studio (Li-Cor) software provided with the Odyssey scanner. Overlay of the fluorescence image with the scored H&E image enabled calling of tumor vs. non-tumor for each grid square.
  • This example describes the evaluation of tumor and adjacent tissues at the cellular level in order to assess sensitivity and specificity of a chlorotoxin conjugate, such as Compound 16 for cancer cells.
  • FIG. 32 shows box & whiskers plots of fluorescence intensity in grid squares from multiple patients and for each tissue section analyzed (T, tumor. NT, adjacent non-tumor tissue. PS, peritumoral skin. S, uninvolved skin). Sensitivity (95%) and specificity (85%) were very good using a threshold of 30,000 (arbitrary units). Peritumoral skin in patient 19 was above this threshold in all grid squares. This patient had an ulcerated tumor and grossly edematous skin immediately adjacent to the mass. The elevated signal in this patient's skin sample accounted for all above-threshold data points in this analysis.
  • Grid squares were called tumor or no tumor based on fluorescence intensity and based on pathologist call. Concordant and discordant calls are used to calculate sensitivity and specificity. The results are shown in Table 50.
  • This example describes the evaluation of tumor and adjacent tissues at the cellular level in order to assess sensitivity and specificity of a chlorotoxin conjugate, such as Compounds 1-720 for cancer cells.
  • Evaluation of canine tumor and adjacent tissues at the cellular level is performed in order to assess sensitivity and specificity of Compounds 1-720 for cancer cells. Sensitivity and specificity are calculated using a grid analysis on 30 micron frozen sections. An overlay of each fluorescence image with the corresponding H&E stained image that was scored as tumor or normal by a histopathologist is analyzed. This analysis is performed separately for each case. Cutaneous squamous cell carcinomas, mammary cancers, and subcutaneous soft-tissue sarcomas are evaluated. Tumor tissue and tissue adjacent to tumors has higher mean fluorescence than does normal tissue.
  • This example describes the use of BLZ-100 for the labeling of other miscellaneous tumor types not described in the preceding examples.
  • Lung cancer is of potential interest clinically.
  • the results of gross imaging in the canine lung cancer suggest specific tumor uptake, with 3:1 TBR compared with adjacent lung or with uninvolved skin.
  • the signal intensity was low but measurable, and was specific for tumor in the primary mass.
  • the suspected metastasis could not be confirmed due to frozen section artifact.
  • Brain tumors are of very high interest for clinical and commercial development of a chlorotoxin conjugate.
  • Intraoperative imaging showed signal in the chlorotoxin conjugate-labeled tumor with a 2.5-fold tumor to background (normal brain) ratio. The surgical approach was through the sinus, so nasal mucosa was present in the image. The mucosal tissues are a known source of background, and in this case provided a positive control for fluorescence intensity. Tumor was removed in fragments; pieces to be analyzed were embedded in OCT and snap-frozen. Imaging of 30 micron sections showed low but detectable signal.
  • the optimal dose for CNS tumors arising within the blood-brain barrier will have to be determined with additional subjects, including cases with malignant tumors such as glioma.
  • this case did provide an opportunity for imaging of normal brain tissue, and it demonstrated that a low-grade brain tumor can be successfully imaged. This is significant, since complete resection in low-grade tumors can be curative.
  • This example describes the use of Compound 16 for the labeling of other tumor types.
  • Intraoperative imaging of a canine meningioma showed signal in the Compound 16-labeled tumor ( FIG. 33A ), with a 2.5-fold tumor to background (normal brain) ratio.
  • the surgical approach was through the sinus, so nasal mucosa was present in the image.
  • the mucosal tissues are a known source of background, and in this case provided a positive control for fluorescence intensity.
  • Tumor was removed in fragments; pieces to be analyzed were embedded in OCT and snap-frozen. Imaging of 30 micron sections showed low but detectable signal ( FIGS. 33B, 33C ). H&E stained sections are shown for comparison ( FIGS. 33D, 33E ). This case demonstrated that a low-grade brain tumor can be successfully imaged.
  • This example describes the use of Compounds 1-720 for the labeling of miscellaneous tumor types, such as mast cell tumors, lung cancer, meningioma, thyroid carcinomas, and oral tumors.
  • miscellaneous tumor types such as mast cell tumors, lung cancer, meningioma, thyroid carcinomas, and oral tumors.
  • Intraoperative tumor imaging shows signals emitted by Compounds 1-720.
  • the tumors are removed in fragments; pieces to be analyzed are embedded in OCT and snap-frozen. Imaging of 30 micron sections shows low but detectable signal. H&E stained sections are used for comparison. Tumors are successfully identified using Compounds 1-720 and optimal doses are determined.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Insects & Arthropods (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
US14/855,355 2013-09-17 2015-09-15 Chlorotoxin conjugates and methods of use thereof Abandoned US20160096869A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US14/855,355 US20160096869A1 (en) 2013-09-17 2015-09-15 Chlorotoxin conjugates and methods of use thereof
US16/704,955 US20200188536A1 (en) 2013-09-17 2019-12-05 Chlorotoxin conjugates and methods of use thereof
US17/409,661 US11559580B1 (en) 2013-09-17 2021-08-23 Tissue-homing peptide conjugates and methods of use thereof
US17/888,072 US20230211000A1 (en) 2013-09-17 2022-08-15 Tissue-homing peptide conjugates and methods of use thereof

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US201361879108P 2013-09-17 2013-09-17
US201361879096P 2013-09-17 2013-09-17
US201461990101P 2014-05-07 2014-05-07
PCT/US2014/056177 WO2015042202A1 (en) 2013-09-17 2014-09-17 Chlorotoxin conjugates and methods of use thereof
US14/855,355 US20160096869A1 (en) 2013-09-17 2015-09-15 Chlorotoxin conjugates and methods of use thereof

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/056177 Continuation WO2015042202A1 (en) 2013-09-17 2014-09-17 Chlorotoxin conjugates and methods of use thereof

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/704,955 Division US20200188536A1 (en) 2013-09-17 2019-12-05 Chlorotoxin conjugates and methods of use thereof

Publications (1)

Publication Number Publication Date
US20160096869A1 true US20160096869A1 (en) 2016-04-07

Family

ID=52689364

Family Applications (2)

Application Number Title Priority Date Filing Date
US14/855,355 Abandoned US20160096869A1 (en) 2013-09-17 2015-09-15 Chlorotoxin conjugates and methods of use thereof
US16/704,955 Abandoned US20200188536A1 (en) 2013-09-17 2019-12-05 Chlorotoxin conjugates and methods of use thereof

Family Applications After (1)

Application Number Title Priority Date Filing Date
US16/704,955 Abandoned US20200188536A1 (en) 2013-09-17 2019-12-05 Chlorotoxin conjugates and methods of use thereof

Country Status (9)

Country Link
US (2) US20160096869A1 (zh)
EP (1) EP3046572A4 (zh)
JP (2) JP6652923B2 (zh)
CN (1) CN105813648A (zh)
AU (4) AU2014323563B9 (zh)
BR (1) BR112016005802A2 (zh)
CA (1) CA2924521A1 (zh)
IL (1) IL244665B (zh)
WO (1) WO2015042202A1 (zh)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017181149A1 (en) * 2016-04-15 2017-10-19 Blaze Bioscience, Inc. Methods of treating breast cancer
WO2017180789A3 (en) * 2016-04-12 2017-11-23 Blaze Bioscience, Inc. Methods of treatment using chlorotoxin conjugates
WO2018119001A1 (en) * 2016-12-19 2018-06-28 Fred Hutchinson Cancer Research Center Peptide-antibody compositions and methods of use thereof
WO2021067563A1 (en) * 2019-10-02 2021-04-08 Blaze Bioscience, Inc. Systems and methods for vascular and structural imaging
US11428689B2 (en) 2016-05-05 2022-08-30 Massachusetts Institute Of Technology Methods and uses for remotely triggered protease activity measurements
US11448643B2 (en) 2016-04-08 2022-09-20 Massachusetts Institute Of Technology Methods to specifically profile protease activity at lymph nodes
US11519905B2 (en) 2017-04-07 2022-12-06 Massachusetts Institute Of Technology Methods to spatially profile protease activity in tissue and sections
US11549951B2 (en) 2009-03-02 2023-01-10 Massachusetts Institute Of Technology Methods and products for in vivo enzyme profiling
US11549947B2 (en) 2011-03-15 2023-01-10 Massachusetts Institute Of Technology Multiplexed detection with isotope-coded reporters
US11559580B1 (en) 2013-09-17 2023-01-24 Blaze Bioscience, Inc. Tissue-homing peptide conjugates and methods of use thereof
US11835522B2 (en) 2019-01-17 2023-12-05 Massachusetts Institute Of Technology Sensors for detecting and imaging of cancer metastasis
US11977074B2 (en) 2013-06-07 2024-05-07 Massachusetts Institute Of Technology Affinity-based detection of ligand-encoded synthetic biomarkers

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6652923B2 (ja) * 2013-09-17 2020-02-26 ブレイズ バイオサイエンス， インコーポレイテッド クロロトキシンコンジュゲート及びその使用方法
CN107847554A (zh) * 2015-06-26 2018-03-27 弗莱德哈钦森癌症研究中心 治疗性肽及其使用方法
EP3442555A4 (en) * 2016-04-15 2020-04-01 Blaze Bioscience, Inc. METHODS OF TREATING BREAST CANCER
RU2638805C1 (ru) * 2016-11-23 2017-12-15 Федеральное государственное бюджетное образовательное учреждение высшего образования "Смоленский государственный медицинский университет" министерства здравоохранения Российской Федерации Способ дифференциальной диагностики аденомы с дисплазией iii степени и ранней аденокарциномы толстой кишки
US11292778B2 (en) 2017-06-05 2022-04-05 The Regents Of The University Of California Heterocyclyl polymethine IR chromophores
CA3086040A1 (en) * 2017-12-19 2019-06-27 Blaze Bioscience, Inc. Tumor homing and cell penetrating peptide-immuno-oncology agent complexes and methods of use thereof
CN109342414B (zh) * 2018-11-22 2021-06-01 江苏师范大学 一种可视化传感阵列芯片及其制备方法
WO2021144450A1 (en) * 2020-01-17 2021-07-22 Fluoguide A/S A composition comprising a fluorophore labelled upar-targeting peptide conjugate
CA3203901A1 (en) 2020-12-30 2022-07-07 Sandor Farkas Chlorotoxin derivatives and use thereof
CN113447583A (zh) * 2021-06-25 2021-09-28 河北冀安检测服务有限公司 一种盐酸氨溴索氯化钠注射液与包材相容性的检测方法
CN113861717A (zh) * 2021-11-09 2021-12-31 西安康福诺生物科技有限公司 一种用于荧光标记的水溶性吲哚菁绿染料及其合成方法
WO2024078579A1 (zh) * 2022-10-12 2024-04-18 江苏亚虹医药科技股份有限公司 吲哚酞菁类化合物、其制备方法及在肿瘤诊断成像中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5968479A (en) * 1995-01-30 1999-10-19 Daiichi Pure Chemicals Co., Ltd. Diagnostic marker
WO2011142858A2 (en) * 2010-05-11 2011-11-17 Fred Hutchinson Cancer Research Center Chlorotoxin variants, conjugates, and methods for their use

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3669752B2 (ja) * 1995-01-30 2005-07-13 第一化学薬品株式会社 診断用マーカー
US8053415B2 (en) * 2005-01-21 2011-11-08 Washington University In St. Louis Compounds having RD targeting motifs
WO2006115633A2 (en) * 2005-04-22 2006-11-02 University Of Washington Cyanine-chlorotoxin conjugate and method for intra-operative fluorescent visualization of cancer
EP2531206B1 (en) * 2010-02-04 2017-05-31 Morphotek, Inc. Chlorotoxin polypeptides and conjugates and uses thereof
AU2011306076B2 (en) * 2010-09-22 2015-05-07 The Board Of Regents Of The University Of Texas System Novel block copolymer and micelle compositions and methods of use thereof
JP6652923B2 (ja) * 2013-09-17 2020-02-26 ブレイズ バイオサイエンス， インコーポレイテッド クロロトキシンコンジュゲート及びその使用方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5968479A (en) * 1995-01-30 1999-10-19 Daiichi Pure Chemicals Co., Ltd. Diagnostic marker
WO2011142858A2 (en) * 2010-05-11 2011-11-17 Fred Hutchinson Cancer Research Center Chlorotoxin variants, conjugates, and methods for their use

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11703510B2 (en) 2009-03-02 2023-07-18 Massachusetts Institute Of Technology Methods and products for in vivo enzyme profiling
US11549951B2 (en) 2009-03-02 2023-01-10 Massachusetts Institute Of Technology Methods and products for in vivo enzyme profiling
US11549947B2 (en) 2011-03-15 2023-01-10 Massachusetts Institute Of Technology Multiplexed detection with isotope-coded reporters
US11977074B2 (en) 2013-06-07 2024-05-07 Massachusetts Institute Of Technology Affinity-based detection of ligand-encoded synthetic biomarkers
US11559580B1 (en) 2013-09-17 2023-01-24 Blaze Bioscience, Inc. Tissue-homing peptide conjugates and methods of use thereof
US11448643B2 (en) 2016-04-08 2022-09-20 Massachusetts Institute Of Technology Methods to specifically profile protease activity at lymph nodes
WO2017180789A3 (en) * 2016-04-12 2017-11-23 Blaze Bioscience, Inc. Methods of treatment using chlorotoxin conjugates
WO2017181149A1 (en) * 2016-04-15 2017-10-19 Blaze Bioscience, Inc. Methods of treating breast cancer
US11428689B2 (en) 2016-05-05 2022-08-30 Massachusetts Institute Of Technology Methods and uses for remotely triggered protease activity measurements
WO2018119001A1 (en) * 2016-12-19 2018-06-28 Fred Hutchinson Cancer Research Center Peptide-antibody compositions and methods of use thereof
US11519905B2 (en) 2017-04-07 2022-12-06 Massachusetts Institute Of Technology Methods to spatially profile protease activity in tissue and sections
US11835522B2 (en) 2019-01-17 2023-12-05 Massachusetts Institute Of Technology Sensors for detecting and imaging of cancer metastasis
WO2021067563A1 (en) * 2019-10-02 2021-04-08 Blaze Bioscience, Inc. Systems and methods for vascular and structural imaging

Also Published As

Publication number Publication date
AU2014323563A1 (en) 2016-04-14
CA2924521A1 (en) 2015-03-26
JP2016534147A (ja) 2016-11-04
AU2021202814A1 (en) 2021-05-27
BR112016005802A2 (pt) 2017-09-12
AU2020200116A1 (en) 2020-01-30
JP6652923B2 (ja) 2020-02-26
CN105813648A (zh) 2016-07-27
US20200188536A1 (en) 2020-06-18
EP3046572A1 (en) 2016-07-27
AU2014323563B9 (en) 2019-10-24
AU2021202814B2 (en) 2023-02-02
IL244665A0 (en) 2016-04-21
WO2015042202A1 (en) 2015-03-26
AU2020200116B2 (en) 2021-02-25
JP2020019779A (ja) 2020-02-06
EP3046572A4 (en) 2017-06-28
AU2014323563B2 (en) 2019-10-10
JP6994011B2 (ja) 2022-02-03
IL244665B (en) 2019-03-31
AU2023202389A1 (en) 2023-07-06

Similar Documents

Publication Publication Date Title
AU2021202814B2 (en) Chlorotoxin conjugates and methods of use thereof
US20230257428A1 (en) Methods of treatment using chlorotoxin conjugates
US20190282661A1 (en) Methods of treating breast cancer
US20240148911A1 (en) FLUORESCENT COMPOUND COMPRISING A FLUOROPHORE CONJUGATED TO A pH-TRIGGERED POLYPEPTIDE
CN108135970B (zh) 软骨归巢肽
US10442890B2 (en) Multifunctional degradable nanoparticles with control over size and functionalities
JP2012520856A (ja) 光学イメージング剤
JP2018521994A (ja) 治療用ペプチドおよびそれらの使用方法
US20230211000A1 (en) Tissue-homing peptide conjugates and methods of use thereof
US7850946B2 (en) Macrocyclic cyanine and indocyanine bioconjugates provide improved biomedical applications
CN107847554A (zh) 治疗性肽及其使用方法
US20210170039A1 (en) Dendrimer compositions and methods for drug delivery to the eye
US20230037660A1 (en) Methods of treating vascular lesions and malformations
EP3442555A1 (en) Methods of treating breast cancer

Legal Events

Date Code Title Description
AS Assignment

Owner name: BLAZE BIOSCIENCE, INC., WASHINGTON

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HANSEN, STACEY J.;JOCHHEIM, CLAUDIA;MILLER, DENNIS M.;AND OTHERS;SIGNING DATES FROM 20151021 TO 20151110;REEL/FRAME:037847/0641

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STCV Information on status: appeal procedure

Free format text: NOTICE OF APPEAL FILED

AS Assignment

Owner name: BLAZE BIOSCIENCE, INC., WASHINGTON

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HANSEN, STACEY J.;JOCHHEIM, CLAUDIA;MILLER, DENNIS M.;AND OTHERS;SIGNING DATES FROM 20190515 TO 20190523;REEL/FRAME:049593/0023

STCV Information on status: appeal procedure

Free format text: APPEAL BRIEF (OR SUPPLEMENTAL BRIEF) ENTERED AND FORWARDED TO EXAMINER

STCV Information on status: appeal procedure

Free format text: EXAMINER'S ANSWER TO APPEAL BRIEF MAILED

STCV Information on status: appeal procedure

Free format text: ON APPEAL -- AWAITING DECISION BY THE BOARD OF APPEALS

STCV Information on status: appeal procedure

Free format text: BOARD OF APPEALS DECISION RENDERED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- AFTER EXAMINER'S ANSWER OR BOARD OF APPEALS DECISION